Polypeptide fragments of hepatitis e virus, the vaccine composition comprising said fragments and the diagnostic kits and the diagnostic kits

ABSTRACT

The present invention relates to polypeptide(s) comprising the amino acid sequence as set forth in SEQ ID No. 1 of hepatitis E virus ORF 2 or its fragment, which is in the form of n-polymeric polypeptide, wherein n is an integer from 1-180; to a chimeric protein consisting of a polypeptide of the present invention and a conserved fragment of hemagglutin antigen from influenza virus; to a polypeptide of the present invention bound to a polypeptide containing epitope from hepatitis E virus ORF3 or an immunogenic fragment thereof; to a recombinant expression vector comprising the DNA molecule encoding the above polypeptides and the host cell transformed with said recombinant expression vector which is able to express polypeptide of the present invention. The present invention further relates to a vaccine composition against hepatitis E virus which comprises the above-mentioned polypeptide, or diagnostic kit for hepatitis E virus infection comprising the above-mentioned polypeptide, which includes IgG, IgM, or total antibody diagnostic kit for hepatitis E virus, and to the use of vaccine composition and diagnostic kit for prophylaxis, diagnosis and/or treatment of hepatitis E virus infection.

FIELD OF THE INVENTION

[0001] The present invention relates to a polypeptide comprising theamino acid sequence as set forth in SEQ ID No. 1 of hepatitis E virusORF 2 or its fragment, which is in the form of N-polymeric polypeptide,wherein N is an integer from 1-180; to a chimeric protein consisting ofa polypeptide of the present invention and a conserved fragment ofhemagglutin antigen from influenza virus; to a polypeptide of thepresent invention bound to a polypeptide containing epitope fromhepatitis E virus ORF3 or an immunogenic fragment thereof; to arecombinant expression vector comprising the DNA molecule encoding theabove polypeptides and the host cell transformed with said recombinantexpression vector which is able to express polypeptide of the presentinvention.

[0002] The present invention further relates to a vaccine compositionagainst hepatitis E virus which comprises the above-mentionedpolypeptide, or diagnostic kit for hepatitis E virus infectioncomprising the above-mentioned polypeptide, which includes IgG, IgM, ortotal antibody diagnostic kit for hepatitis E virus, and to the use ofvaccine composition and diagnostic kit for prophylaxis, diagnosis and/ortreatment of hepatitis E virus infection.

BACKGROUND OF THE INVENTION

[0003] Hepatitis B Virus (HEV) was firstly recognized as a pathogen toenterically transmitted non-A, non-B hepatitis in 1983 (Balayan et al.,1983. Intervirology 20:23). Hepatitis E is mainly endemic in developingcountries in Asia, Africa and Middle America. In developed countries,hepatitis E cases were mostly found in immigrants or traveler fromabroad. Both sporadic cases and large epidemic have been documented.During the period from 1950s to 1990s, several hepatitis E outbreakshappened sequentially due to polluted drinking water(Visvanathan, 1957.Indian J. Med. Res.(Suppl.). 45:1-30; Wong et al., 1980 Lancet.,2:882-885; Myint et al., 1985, Am J Trop Med Hyg., 34:1183-1189;Belabbes et al., 1985 J Med Virol., 16:257-263; Hau et al., 1999, Am JTrop Med Hyg., 60:277-280). Most hepatitis E infection was self-limitedand scarcely developed into chronic; but for the pregnant, the sequelwas severe with a mortality rate above 17% (Tsega et al., 1992, Clin.Infec Dis., 14:961-965; Dilawari et al., 1994, Indian J Gastroenterol.,13:44-48; Hussaini et al., 1997, J Viral Hepat., 4:51-54).

[0004] In 1991, researchers got the first complete genome sequence ofHEV, a single-strand non-enveloped positive RNA virus (Tam et al., 1991,Virology 185:120-131 ). Sequence analysis showed the genome was 7.2 kbwith three open reading frames. ORF1 which locates at 5′ end encodesnon-structural protein of the virus, ORF2 which locates at 3′ endencodes major structural protein of the virus. At ORF3 5′ end, there isone bp overlapped with ORF1 3′ end. At ORF3 3′ end, there are 339 bpoverlapped with ORF2 end. It's acknowledged that ORF3 encodes anotherstructure protein with unknown function (Tam et al., 1991, Virology,185:120-131; Aye et al., 1992, Nucleic Acids Res., 20:3512; Aye et al.,1993, Virus Genes., 7:95-109; Huang et al, 1992, Virology, 191:550-558;Reyes et al., 1993, Arch Virol Suppl.,7:15-25)

[0005] The detection of HEV infection mainly depended on ImmunologicalElectron Microscope (IEM) or Immunological Fluorescence Technique for along time, but those techniques are very complicated, expensive and hardto be fulfilled in many laboratories. After the clone and sequencing ofHEV genome, more sensitive techniques like ELISA, Western Blot, PCR,etc. were developed to be used in the detection of HEV infection.

[0006] It is well recognized that the development of serum HEV antibodykits is absolutely necessary, but due to very low-concentration HEVvirus secreted by the infected human or animals, thus it is impossibleto use sera as the source of antigen. Till now, the efficiency of HEVcell culture is still very low, which limited the availability of enoughantigens for HEV detection. Thus, the detection of HEV antibody is stilldepending on synthesized polypeptides or recombinant antigens.Unfortunately, many serological studies showed greatly variedconsistence based on synthesized polypeptides or recombinant antigensfrom different HEV genome regions. For example, Goldsmith et al., (1992,Lancet, 399:328-331 )used ORF2 3-2(M)antigen(a.a. 613-660, Mexicostrain)to detect in-hospital hepatitis E virus infection cases. Thedetecting rate of IgG was 91%, and fell to 27˜50% after 6˜12 month. Whenhe used 3-2(B) (The same ORF2 fragments from Burma strain) in detection,the rate was just 64% and no positive result was found after 6˜12months. On the contrary, 3-2(M) could not react with the convalescentserum in a Parkistan subject, but 3-2(B) could react with the serum ofthe same case 4.5 years later. For those proteins, when the antibody insome cases turned negative, in others it still remained in high titer.The results were similar when the mosaic protein with several linearepitopes expressed in E. Coli was used. Lack of good HEV antibody kitslimited deep research on the dynamic of antibody during HEV infection.In general, during HEV infection, specific IgG antibody is detectable inearly stage, peaks after 2-4 weeks and declines quickly. Most turned tonegative after 9 months, but some patients kept positive many yearslater. Recently, several recombinant antigens have been expressed inboth baculoviras and E. coli, which reacts strongly with sera from bothacute and convalescence phase. In principle, these antigens are moresuitable for sero-epidemiological survey: the titer of serum HEV IgGfell rapidly after acute stage, but it still retained at a detectablelevel. It's worthy of notification that the antibody is related to theprotection from infection during the disease epidemic.

[0007] In the other way, due to the fact that only indirect methods ofdetecting methods are available up to date, no established HEV IgM kitis ever developed around world. With respect to the indirect methods, onthe one band, the detect result is affected by various factors andreproduction is poor; on the other, the reliability of the result ispoor for high possibility of false positive, higher negative value, orlower sensitivity. According to recent reports, during the detection ofclinical samples, when the result for IgM is positive, IgG is generallypositive too, thus its value on early diagnosis is limited bat may behelpful in elevation of the specificity of diagnosis of acute infection.

[0008] It's reported that the antibody to several synthesized peptideand some recombinant antigens will disappear quickly in many infectedsubjects, so clinical diagnosis on acute hepatitis E virus infection isgenerally based on IgG antibody with a higher clinical concordance, butthe most important defect of this method is that it can not distinguishpast infection from recent infection, which will lead to both falsediagnosis in high hepatitis E virus endemic area and under evaluation ofthe prevalence of hepatitis E virus infection during epidemiologicalstudies. Therefore, there is urge demand to develop a reliable andsensitive anti-μ chain IgM diagnostic kit and IgG diagnostic kit whichis characterized by high sensitivity toward convalescence sera.

[0009] In recent years, there was some progress in developing a highlysensitive IgG diagnostic kit. Mast et al. (1998, Hepatology,27: 857-861)provided a comprehensive evaluation of 10 major IgG antibody EIAs aroundworld. The concordance of many kits was fairly good in detecting knownpositive sera, but great difference existed among different kits indetecting American blood donors. It implied that the reliability of theresults is worse in HEV prevalence studies in non-endemic areas. Amongthose kits, most antigens are based on linear epitopes of HEV, but twokits used conformational epitopes as antigens. First is ORF2.1(aa394˜660), the other is baculovirus expressed VLP (aa112˜607). Bothantigens can detect convalescence antibody, but direct data on thecomparison of the concordance between those two antigens is notavailable till now. It's possible that those two antigens identifieddifferent antibodies. In addition, nearly 20% prevalence was reported innon-endemic America using VLP kit, which aroused the suspicion of itsspecificity. But with the reported positive HTV infection in swine,goats, cows, chickens, rats, wild monkeys and enclosed monkeys, togetherwith separately 77% and 44% antibody prevalence among wild rats inMaryland and Louisanna, it's possible that the antibody prevalence isunderestimated in American population, though animal HEV can not causeclinical disease due to its virulence. (Kabrane-Lazizi et al., 1999 Am JTropic Medicine, 61:331-335). And ORF2.1 kit can detect higher positiverate among CMV infection and autoimmunological diseases. In addition,the reported ORF2.1 polypeptide, which is a GST-conchimeric protein orpolyarginine conchimeric protein, intends to obtain false positiveresults in practice.

[0010] Both cell and tissue culture for HEV have ever been successful,and practical methods to get a large amount of virus is not yetavailable, so it's the only research way to switch from tradition killedor attenuated vaccine to subunit or DNA vaccine through geneticengineering.

[0011] HEV ORF2, beginning at the base positioned at 5147, has 1980nucleotides, which encodes a polypeptide with 660 amino acids presumedto be major structural protein and constitutes the capsid of virus. At Nterminal of ORF2 protein, there is a classical signal sequence followedby a region rich in arginine, which is highly positive charged regionand believed to involve in genomic RNA encapisidation during virusassembly. During translation process, ORF2 entered endoplasmic reticulum(ER) by a mechanism of signal peptide recognized protein (SRP), and isfurther glycosylated and accumulated in ER, then probably formed thecapsomer of capsid in suit. Three N-glycosylated sites, Asn-137, Asn-310and Asn-562, are located at ORF2. They are highly conservative amongdifferent virus strains, and Asn-310 is the major glycosylated site.ORF2-transfected mammalian cell COS, human hepatocellular carcinomaHuh-7, HepG2 can thereby express a 88 kD glycoprotein which can be foundin both cytoplasma and membrane. The mutation in those glycosylatedsites did not affect the location of PORF2 onto cell membrane. Howeverafter the signal peptide sequence was removed therefrom, PORF2 can onlybe found in cytoplasma. This implied that the shift of PORF2 instead ofglycosylation is necessary to protein location onto cell membrane. LikeMS protein in HBV, PORF2 is possibly secreted to cell membrane directlythrough ER instead of Golgi body. On the surface of transfected cell,gpORF2 is not randomly distributed, but concentrated in some zone, whichimplied an active combination process of a protein subunit and maybeaggregate into some more ordered advance forms. The finalassembly/maturation of the virus need the encapisidation of genomic RNA,thus it must be occurred in cytoplasma outside of ER or endo-wall ofcell membrane. The accumulation of gpORF2 in membrane may imply theassembly of virus. At the same time, the location of capsid protein onmembrane also implied the possibility of secretion of matured virus outof the cell through budding. One more attention should be drawn that,the in vitro transcript and translation of PORF2 using in-vitrotranslation system with translating and modifying function can produce88 kD of gpORF2 in forms of both monomers and dimmers. It illustratedthat gpORF2 was prone to form homologous dimmer, and capsomer of capsidmay be constituted by said homnologuos dimer of gp ORF2 (Jameel et al.,1996. J. Virol., 70:207-216.). Through Frost Etching electionmicroscope, Li et al. found that recombinant HEV VLPs which is expressedby baculovirus had an icosahedral symmetry virous (T=1), which was madeup of sixty p50 subunits with 22-23 nm in diameter. Since the innerspace of this size particle can contain about 1 kb RNA, and HEV genomeis 7.5 kb in length, it is speculated natural HEV should be a crystallattice structure with T≧3, but the topological structure of capsomer issimilar. The total number of T=3 subunit is 90 capsomers (Li et al.,1999. virology, 265:35-45.)

[0012] According to the above, HEV is a non-enveloped virus. Viruscapsid is made up of ORF2-encoded protein. The protein embodies majorimmunological epitopes and some neutralizing epitopes, thus it becamethe most favorable fragment during subunit vaccine research.

[0013] In U.S. Pat. No. 5,885,768, Reyes et al. firstly reported that 4cynomolgus monkeys were injected in with recombinant protein trpE-C2expressed in E. Coli comprising HEV Burma strain ORF2 C terminal ⅔(aa225˜660), wherein said protein is formulated with an alum adjuvant,by administering at 0, 30 day for 50 μg/dose. Another 2 monkeys wereused as controls with adjuvant only. Four weeks later, no positiveresult regarding raised antibody from collected bloods is found byWestern Blotting. A third-time immunization on two monkeys among them byadministering 80 μg unsolvable recombinant protein without adjuvant.Four weeks later, both monkeys were positive (WB). Then the six monkeyswere grouped into first and second group, each included three monkeys,two of them is immunized with either three-times or two-timesinoculation, and one is control. The first group was attacked with BurmaHEV, and the second group Mexico HEV. The results were, (1) ALT keptnormal all the time in the immunized group, but it increased 6˜10 timeshigher than before immunization in control; (2) Liver biopsy sample wasdetected by Immunological Fluorescence method. The antigen can be foundin all other monkeys except those immunized with three doses andattacked by Burma strain. (3) Virus excretion in feces can be foundsequentially in all other monkeys except those immunized with threedoses and attacked by Burma strain. This research sample is small, butit implied that recombinant protein from ORF2 can block the occurrenceof biochemical indexes of virus hepatitis and protected completely frominfection when the monkeys were attacked by wild HEV.

[0014] Tsarcv et al. (1994, Proc. Nat. Acad. Sci. USA., 91:10198-10202:Tsarev et al., 1993, J. Infect. Dis., 168:369-378: Tsarev et al., 1997,Vaccine 15:1834-1838) used baculovirus in insect (SF cell) to expressHEV ORF2 and got protein particles with various size from 20 nm-30 nm incell solution. The percentage of smaller particles is substantivelyincreased during anaphase of infected cells. WB method was used todetect baculovirus expressed ORF2 with many specific different-sizebands at 25 kD, 29 kD, 35 kD, 40˜45 kD, 55˜70 kD, 72 kD. Ion exchangeand molecular screening method were used to purify HEV specific protein.One day after recombinant virus infected the cells, the whole ORF2peptide of 72 kD firstly appeared and then disappeared gradually. On thesecond day, the peptides of 63 kD and 55 kD appeared. On the first day,53 kD peptide appeared in cell solution with large amount and peaked onthe third day. This implied the primary 72 kD protein was randomly cutinto HEV protein with 55 kD (in cell lysis solution) and/or 53 kD (cellsolution). The sequencing to those two proteins showed 55 kD located atORF2 aa112˜607, but 53 kD located at aa112˜578 and 63 kD at aal 126˜660.The results of ELISA showed the activity of 55 kD was apparentlystronger than 53 kD. If aa112˜660 fragment was expressed in baculovirusin insect, 63 kD and 55 kD recombinant HEV protein can also be found.

[0015] SF9 cells were collected at day 7 from the cells had beeninfected. The protein was primarily purified and absorbed with alumadjuvant. Then cynomolgous monkeys were immunized i.m. with 50 μgprotein per dose. Four received 1 dose, the other four two doses (0 d,28 d). After the final dose, all monkeys were attacked with dose1000˜1000CID50 i.v. of the same HEV strain (SAR-55, from a Parkistanpatient). Within 15 weeks, liver biopsy, sera and feces were collectedevery week. Before virus attack, antibody titers in one-dose monkeyswere 1:100˜1:10000, but in two-dose group they were all 1:10000 (coatedwith purified 55 kD). In one-dose group, one monkey was dead due toanesthesia accident 9 weeks after virus attack (still calculated in theresults). In two-dose group, two monkeys died soon after the virusattack (no calculated in the results) due to unknown reason. Six monkeysafter immunization were found no ALT elevation or liver biopsypathological change, and no viremia. Among four monkeys in one-dosegroup, three has virus excretion, but two monkeys in two-dose group novirus excretion was found.

[0016] Further purification was done in 55 kD protein expressed inbaculovirus system through ion exchange and molecular sifting methods tomake its purification reachable above 99%. After absorbed with alumadjuvant, the protein with dose 50 μg, 10 μg, 2 μg, 0.4 μg, 0μg(control)was each injected into 4 rhesus monkeys administered 0 and 28day. Four weeks after the last dose, the monkeys were attacked with thesame virus (SAR-55). Sixteen monkeys in the immunized group were allnormal, and just one monkey with 2 μg dose and the other with 0.4 μgdose appeared very light pathological change. Though the immunized canprevent from hepatitis but not infection. All sixteen monkeys immunizedappeared virus excretion, also viremia except one monkey with 50 μg doseand the other with 10 μg dose. And the amount of virus was limited inmost monkeys, but the duration has not been shortened. Another fourmonkeys were immunized with 2×50 μg, and attacked with 100,000 MID50other HEV 4 weeks after the final dose. The results were similar. Allfour monkeys did not show ALT elevation and pathological change, butonly one monkey did not show virus excretion and viremia. The amount ofvirus decreased apparently, but the duration has not been shortened.According to the author's opinion, the effect of complete protection onthose monkeys was worse that previous time. It's possibly attributableto the amount of virus used in attack. The amount of virus in thisexperiment reached 300,000, but was 1000˜10000MID50 last time. One more,the titer of antibody among groups from 0.4 μg to 50 μg has showed nodifference before attack.

[0017] The staffs in Genelabs company expressed ORF2 aa112˜660 using thesame baculovirus in insect and got a large amount solvable recombinant62 kD protein. After purification, the cynomolgous monkeys wereimmunized and protected from the attack of virus (Mexico strain) withdose 1000CID50(3 monkeys immunized with 20 μg did not get disease. Virusexcretion was not found in two monkeys, and the amount of virusexcretion decreased in one monkey). (Zhang et al., 1997, Clin Diagn LabImmunol.;4:423-8.)

[0018] McAtee et al., (1996, Protein Expr. Purif., 8:262-270) preparedBurma ORF2 62 kD) dimerexpressed in recombinant baculovirus. Thedimerwas dissociated into two peptides separately with 56.5 kD and 58.1kD through HPLC-MS. Peptide mass fingerprint analysis showed the Nterminal of those two peptides was the same aa112, and the C terminal isseparately aa637 and aa652. And 56.5 kD protein was a very goodimmunogen.

[0019] Anderson group in Australia (Anderson et al., 1999. J. Virol.Methods., 81:131-142; Li et al., 1994, J Clin Microbio.)32:2060-2066: Liet al., 1997 J. Med. Virol., 52:289-300: Li et al., 2000, J. Med.Virol.,. 60:379-386) used ORF2 aa394˜660 (ORF2.1) expressed in E. Coli.The product is a GST-conchimeric or poly arginine protein which can forma highly conformational convalescence epitope. This epitope can detect avery high-rate convalescence sera, but it will disappear when thefragment was extended or truncated towards N terminal. The serum at 30weeks after rats were immunized with recombinant ORF2.1 protein was usedto block the serum from convalescence patients with VLP expressed inbaculovirus as the coated antigen. The blocking rate will reach 81%˜86%.Monoclonal antibody was prepared with ORF2.1 protein and two monoclonalantibody 2E2 and 4B2 which can identify ORF2.1 conformational epitopes,and five possible identifiable monoclonal antibodies were obtained. Theblocking rate can reach 60% whether 2E2 or 4B2 was used to blockconvalescence sera with VLPs as antigen. This implied that those twomonoclonal antibodies can identify the epitopes which was majorcomponents of antibody identified epitopes in convalescence sera.Different data showed that ORF2.1 had major epitope structure rathersimilar to VLP. The antibody to the epitopes can exist for a long timein HEV infected serum. It's probably an important protective epitope,but animal protection experiment about ORF2.1 has not been reported tillnow.

SUMMARY OF THE INVENTION

[0020] In one aspect of the present invention, it provides a polypeptidecomprising the amino acid sequence of hepatitis E virus open readingframe (ORF) 2 (as set forth in SEQ ID No. 1) or its fragment, which isin the form of n-polymeric polypeptide, wherein n is an integer from1-180, said polypeptide comprising the amino acid as set forth in SEQ IDNo. 1 of hepatitis E virus ORF 2 or its fragment is selected from thegroup consisting of:

[0021] 1) A polypeptide having the amino terminus starts from betweenamino acid residues 113 and 469, and the carboxyl terminus ends frombetween amino acid residues 596 and 660;

[0022] 2) A polypeptide having the amino terminus starts from betweenamino acid residues 370 and 469, and the carboxyl terminus ends frombetween amino acid residues 601 and 628;

[0023] 3) A polypeptide having the amino terminus starts from betweenamino acid residues 390 and 459, and the carboxyl terminus ends frombetween amino acid residues 601 and 610;

[0024] 4) A polypeptide having the amino acid sequence of amino acidresidues 414 to 660 from SEQ ID NO: 1, i.e., polypeptide 247;

[0025] 5) A polypeptide having the amino acid sequence of amino acidresidues 429 to 660 from SEQ ID NO: 1, i.e., polypeptide 232;

[0026] 6) A polypeptide having the amino acid sequence of amino acidresidues 439 to 660 from SEQ ID NO: 1, i.e., polypeptide 222;

[0027] 7) A polypeptide having the amino acid sequence of amino acidresidues 459 to 660 from SEQ ID NO: 1, i.e., polypeptide 201;

[0028] 8) A polypeptide having the amino acid sequence of amino acidresidues 394 to 628 from SEQ ID NO: 1, i.e., polypeptide 235N;

[0029] 9) A polypeptide having the amino acid sequence of amino acidresidues 394 to 618 from SEQ ID NO: 1, i.e., polypeptide 225N;

[0030] 10) A polypeptide having the amino acid sequence of amino acidresidues 394 to 602 from SEQ ID NO: 1, i.e., polypeptide 209N;

[0031] 11) A polypeptide having the amino acid sequence of amino acidresidues 394 to 601 from SEQ ID NO: 1, i.e., polypeptide 208N;

[0032] 12) A polypeptide having the amino acid sequence of amino acidresidues 394 to 606 from SEQ ID NO: 1, i.e., polypeptide NE2I;

[0033] 13) A polypeptide having the amino acid sequence of amino acidresidues 390 to 603 from SEQ ID NO: 1, i.e., polypeptide 217D;

[0034] 14) A potypeptide having the amino acid sequence of amino acidresidues 374 to 618 from SEQ ID NO: 1, i.e., polypeptide 205;

[0035] 15) A polypeptide having the amino acid sequence of amino acidresidues 414 to 602 from SEQ ID NO: 1, i.e., polypeptide 189;

[0036] 16) A polypeptide having the amino acid sequence of amino acidresidues 414 to 601 from SEQ ID NO: 1, i.e., polypeptide 188;

[0037] 17) A polypeptide having the amino acid sequence of amino acidresidues 459 to 628 from SEQ ID NO: 1; and

[0038] 18) A polypeptide having the amino acid sequence of amino acidresidues X to 603 from SEQ ID No: 1 with Met added at N-terminus and amodified C-terminus, wherein said modified C-terminus refers to add, inthe direction from 5′-3′, amino acid sequence -Pro-Pro-Arg at amino acidresidue 603, Pro, on its 3′ end; including:

[0039] a) when X is amino acid residue 394, said polypeptide is as setforth in SEQ ID NO: 2, i.e., NE2;

[0040] b) when X is amino acid residue 414, said polypeptide is as setforth in SEQ ID NO: 3, i.e., 193C;

[0041] c) when X is amino acid residue 429, said polypeptide is as setforth in SEQ ID NO: 4, i.e., 178C;

[0042] d) when X is amino acid residue 439, said polypeptide is as setforth in SEQ ID NO: 7, i.e., 168C;

[0043] e) when X is amino acid residue 449, said polypeptide is as setforth in SEQ ID NO: 8, i.e., 158C;

[0044] f) when X is amino acid residue 459, said polypeptide is as setforth in SEQ ID NO: 9, i.e., 148C;

[0045] g) when X is amino acid residue 469, said polypeptide is as setforth in SEQ ID NO: 10, i.e., 138C;

[0046] In another aspect of the present invention, it further provides apolypeptide having at least 80% homology to any one of the precedingpolypeptides as presented in the above 1)-18) and having substantiallyidentical biological property, such as antigenicity or immunogenicity,etc.

[0047] In another aspect of the present invention, it further provides arecombinant expression vector comprising the nucleotide sequenceencoding the above-mentioned polypeptides of the present invention. Inanother aspect of the present invention, it further provides a host celltransformed with any one of the above recombinant expression vectors,which is able to express the polypeptide(s) of the present invention.

[0048] In another aspect of the present invention, it further provides avaccine composition for prophylaxis and/or treatment of hepatitis Evirus infection in mammals, which comprises at least one of thepolypeptides of the present invention or any combination thereof, andoptionally, pharmaceutically acceptable vehicles and/or adjuvant.

[0049] In another aspect of the present invention, it further provides achimeric protein comprising a polypeptide of the present invention and aconserved fragment of hemagglutin antigen from influenza virus.

[0050] In another aspect of the present invention, it ether provides avaccine composition for prophylaxis and/or treatment of hepatitis Bvirus infection in mammals, which comprises chimeric protein consistingof one of polypeptides of the present invention and a conserved fragmentof hemagglutin antigen from influenza virus, and optionally,pharmaceutically acceptable vehicles and/or adjuvant.

[0051] In another aspect of the present invention, it further providesuse of the above-mentioned vaccine compositions for vaccinating mammalsto prevent from hepatitis E virus infection.

[0052] In another aspect of the present invention, it further provides amethod for prophylaxis and/or treatment of hepatitis E virus infectionin mammals, which comprises administrating to the subject with aprophylaxis and/or treatment effective amount of at least one of theabove-mentioned polypeptide (s) or chimeric protein (s) consisting of atleast one of the above-mentioned polypeptide and a conserved fragment ofhemagglutin antigen from influenza virus.

[0053] In another aspect of the present invention, it further provides adiagnostic kit for the diagnosis of hepatitis E virus infection inbiological sample, which comprises a diagnosis effective amount of atleast one of the polypeptides of the present invention or anycombination thereof.

[0054] In another aspect of the present invention, it further provides adiagnostic kit for the diagnosis of hepatitis E virus infection inbiological sample, which comprises a diagnosis effective amount of atleast one of the polypeptides of the present invention or anycombination thereof, and further comprises the polypeptide containingimmunogenic epitope from hepatitis E virus ORF3 or an immunogenicfragment thereof, wherein said polypeptide containing immunogenicepitope from hepatitis E virus ORF3 or an immunogenic fragment thereofis, optionally, covalently bound to said polypeptide.

[0055] In another aspect of the present invention, it further provides amethod for diagnosis hepatitis E virus infection in biological samples,comprising contacting the above-mentioned diagnostic kit with sample tobe detected under the conditions suitable for the interaction of antigenand antibody.

[0056] In another aspect of the present invention, it further provides amethod for detecting total antibodies against hepatitis E virus, amethod for detecting antibody IgG against hepatitis E virus, and amethod for detecting antibody IgM against hepatitis B virus inbiological samples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057]FIG. 1 presents a schematic diagram of the construction of plasmidpTO-T7-ORF2-201 for expression of the polypeptide 201.

[0058]FIG. 2 shows the results of analysis by 12% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) (Coomassie brilliant blue R250 staine)regarding culture lysates of the induced E. coli transformed withexpression vector pTO-T7-ORF2-201 (with steps of: centrifuging culturemedium, collecting precipitated cell, then resuspending pellet withloading buffer including 0.1% SDS, further treating it in boiling water10 min, then centrifuging under 12,000 rpm for 10 min, takingsupernatant for determination). Lanes 1 and 2 respectively contain twodifferent bacteria culture lysates. Expressed products take up around35% of total protein as analyzed by Uvi gel imaging system (UVItec,ltd., model DBT-08).

[0059]FIG. 3 shows the analysis results of Coomassie blue R250-stained12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) regarding 2Mand 4M Urea solution of purified polypeptide 201 inclusion body fromfour batches of polypeptide 201, wherein said samples are obtained fromrecombinant E. coli embodying expression vector pTO-T7-ORF2-201. Theresults show that some of the polypeptide 201 had undergon renaturationto form dimmer polypeptide with a proportion for the dimmer polypeptidevaried from 10% to 60%. The proportion for renatiation is lower thanthat of sample renatured in 1×PBS (20×PBS (1L):Na₂HPO₄-12H₂O, 73.344 g;KH₂PO₄, 4 g; NaCl, 163.632 g; KCl, 4.024 g, pH7.45). As shown in FIG. 4,percent of dimmer is 99%.

[0060]FIG. 4 shows the results of Western blotting analysis ofpolypeptide 201 with serum from HEV-infected patient. Lanes 1-3 is SDSPAGE control, wherein lane 1, protein molecular weight marker; lane 2,renatured sample of polypeptide 201 in 1×PBS; Lane 3, renaturedpolypeptide 201 treated in boiling water bath for 10 min; Lane 4 and 5,the respective Western blot results corresponding to the sample of Lanes2 and 3.

[0061]FIG. 5 shows the results from hydrated dynamic semi-diameter bydynamic light scattering instrument of aforementioned polypeptide 201,wherein polypeptide 201 is in advance purified by gel filtration HPLC,and centrifuged for 10 min under 20000 g, filtrated with 0.1 um filtermembrane.

[0062]FIG. 6 shows Western blot results of the reaction of polypeptide208N, 209N and 225N with mouse Mab 1F6, 2C9 and 3F5. Lanes 1, 2, 3,respectively, corresponds to the renatured sample being treated inboiling water bath for 10 min, renatured sample and the precipitatedrenatured samples of polypeptide 208N; Lanes 4, 5, 6, respectively,corresponds to the renatured sample being treated in boiling water bathfor 10 min renatured sample and the precipitated renatured samples ofpolypeptide 209N; Lanes 7, 8, 9, respectively, corresponds to therenatured sample being treated in boiling water bath for 10 min,renatured sample and the precipitated renatured samples of polypeptide225N; and Lane 10 is monomer polypeptide 201 as control.

[0063]FIG. 7 illustrates the profile of HEV antibodies raised in serafrom mice following immunization with vaccine of polypeptide 201(containing Feund's adjuvant) in various dosages. The horizontalcoordinate is defined as the days after the first immunization. Thevertical coordinate is defused as the OD_(450 nm/620 nm) measured byELISA.

[0064]FIG. 8 illustrates the profile of HEV antibodies raised in serafrom mice following immunization with vaccine of polypeptide 201(containing no adjuvant) in various dosages. The horizontal coordinateis defined as the days after the first immunization. The verticalcoordinate is defined as the OD_(450 nm/620 nm) of ELISA.

[0065]FIG. 9 illustrates the profile of HEV antibodies raised in serafrom mice following immunization with vaccine of polypeptide 201(containing aluminum hydroxide as adjuvant) in various dosages. Thehorizontal coordinate is defined as the days after the firstimmunization. The vertical coordinate is defined as theOD_(450 nm/620 nm) of ELISA.

[0066]FIGS. 10A, 10B, 10C and 10D show the profile of HEV antibodiesraised in sera from rhesus monkeys grouped No.1, No.2, No.3 and No.13,respectively. All these subjected animals are challenged with HEV byintravenous injection. The horizontal coordinate is defined as the daysafter the first immunization. The vertical coordinate is defined as theOD_(450 nm/620 nm) of ELISA. The results illustrate the anti-NE2I-IgG ispresent 5-10 days earlier than GENELABS-IgG, and WANTAI anti-HEV-IgG insera of monkeys in group No.1, No.2, and No.3; the anti-NE2I-IgG isdetectable in sera of No.13 monkeys, and no Genelabs anti-HEV-IgG andWANTAI anti-HEV-IgG can be detected in sera of NO.13 monkeys.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0067] Unless otherwise indicated, all the terms or nomenclatures usedherewith are the same as those conventionally used in the art. Theconventional manufactures in cell culturing, molecular genetics, nucleicacid chemistry, and immunological procedure are carried out as routinetechnique in the art. In the present invention, unless otherwiseindicated, these terms used herewith have the meanings as follows:

[0068] “hepatitis E virus” or “HEV” refers to a virus, virus type orvirus class, which i) causes water-borne, infectious hepatitis; ii) itdistinguishes from hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), or hepatitis D virus (HDV) in terms ofserological characteristics; iii) contains a genomic region that ishomologous to a 1.33 kb cDNA inserted in pTZKF1(ET1.1), said plasmid isembodied in a E. coli strain deposited in American Type CultureCollection (ATCC) with accession number 67717.

[0069] The Polypeptide of the Present Invention

[0070] In one aspect, the present invention surprisingly provides aseries of polypeptide fragment of HEV with satisfied antibody reactivityand/or immunogenicity, wherein said fragment is included in the aminoacid sequence of HEV ORF2, as set forth in SEQ ID NO: 1. The name ofindividual fragment could be found in table 1 of example 6.

[0071] In the present invention, the numbering of the amino acid residueby position in the amino acid sequence is in accordance with thenumbering manner of International Union of Pure and Applied chemistryand International Union of Biochemistry, Joint commission on biochemicalNomenclature, “Nomenclature and symbolism for Amino Acids and Peptides”,Pure Appl. Chem., 56, 595-624 (1984). Specifically, the coding startsite Met in Seq Id No: 1 is designed as position 1, increased in thedirection from 5′ to 3′.

[0072] In one aspect of the present invention, a polypeptide isprovided, which comprises the amino acid sequence as set forth in SEQ IDNo. 1 of hepatitis E virus ORF 2 or its fragment in the form ofn-polymeric polypeptide, wherein n is an integer from 1-180. When n is2, said polypeptide is a dimmer polypeptide; when n is 3, saidpolypeptide is a trimmer polypeptide; when n is 4, said polypeptide is atetrameter polypeptide, and so on.

[0073] In the present invention, the amino terminus (5′ end) of saidpolypeptide fragment comprising amino acid sequence as set forth in SEQID NO: 1 starts from between amino acid residue 113 and 469, preferably,from between amino acid residue 370 and 469, more preferably, frombetween amino acid residue 390 and 459; and the carboxyl terminus (3′end) of said poly peptide ends from between amino acid residues 596 and660, preferably, from between amino acid residue 601 and 628, morepreferably, from between amino acid residue 601 and 610. Specifically,the preferable polypeptides of the present invention are polypeptide247, polypeptide 232, polypeptide 222, polypeptide 201, polypeptide235N, polypeptide 225N, polypeptide 209N, polypeptide 208N, polypeptideNE2I, polypeptide 217D, polypeptide 205, polypeptide 189, polypeptide188, and the polypeptide having amino acid sequence from amino acidresidue 459 to 628 of SEQ ID NO; 1.

[0074] In the present invention, it further relates to the polypeptidehaving the amino acid sequence of amino acid residues X to 603 from SEQID NO: 1 with Met added at N-terminus and a modified C-terminus, whereinsaid modified C-terminus refers to add, in the direction from 5′-3′,amino acid sequence -Pro-Pro-Arg at amino acid residue 603, Pro, on its3′ end; including: a) NE2; b) 193C; c) 178C; d) 168C; e) 158C; f) 148C;g) 138C.

[0075] In another aspect of the present invention, it further relates toa polypeptide having at least 80% homology to any one of the precedingpolypeptides and having substantially identical biological property,such as antigenicity or immunogenicity, i.e., the derivates of thepolypeptide of the present invention. Specifically, the polypeptide isconsidered as derivates of polypeptide of present invention under thecondition that the amino acid of said polypeptide comprises the aminoacid sequence of aforementioned polypeptide with other amino acid than anatural sequence neighboring to the present polypeptide at N-terminusand/or C-terminus thereof, but it still substantively remain the similarbiological property, such as, antigenicity and or immunogenicity etc. tothe present polypeptide. Consequently, DNA fragment corresponding to thesame is called derivate DNA of present invention. For example, for thepurpose of expression and/or purification, it would be facilitate topurification by adding start amino acid (Methionine) or other leadingpeptide and/or signal peptide at N-terminus therof, or by adding severalHistidines at C-terminus thereof.

[0076] The present invention further contemplates the polypeptide havingidentical biological property, such as antigenicity and/orimmunogenicity, etc. to any one of aforementioned polypeptides; orpolypeptide having at least 80% sequence identity to any one ofaforementioned polypeptides. The term “percentage identity” is intendedto denote a percentage of nucleotides or of amino acid residues whichare identical between the two sequences to be compared, obtained afterthe best alignment, this percentage being purely statistical and thedifferences between the two sequences being distributed randomly andover their entire length. Sequence comparisons between two nucleotide oramino acid sequences are conventionally carried out by comparing thesesequences after having aligned them optimally, said comparison beingcarried out be segment or by “window of comparison” in order to identifyand compare local regions of sequence similarity. The optimal alignmentof the sequences for comparison may be produced, besides manually, bymeans of the local homology algorithm of Smith and Waterman (1981) AdsApp. Math., 2:482, by means of the local homology algorithm of Neddlemanand Wunsch (1970) J. Mol. Biol., 48:443, by means of the similaritysearch method of Pearson and Lipman (1988), Proc. Natl Acad. Sci., USA,85: 2444, by means of computer programs which use these algorithms (GAP,BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.).

[0077] The percentage identify between two nucleic acid or amino acidsequences is determined by comparing these two sequences aligned in anoptimal manner in which the nucleic acid or amino acid sequence to becompared may comprise additions or deletions compared to the referencesequence for optimal alignment between these two sequences. Thepercentage identity is calculated by determining the number of identicalpositions for which the nucleotide or the amino acid residue isidentical between the two sequences, dividing this number of positionscompared and multiplying the result obtained by 100 so as to obtain thepercentage identity between these two sequences.

[0078] For example, program BLAST “BLAST 2 sequences” can be used whichis obtained from website http;//www.ncbi.nlm.nih.gov/gorf/b12.html,wherein parameter is default (particularly, open gap penalty is 5,extension gap penalty is 2; matrix is provided by such as program“blosum 62”), the percentage of identity between two sequences to bealigned is directly calculated by the program.

[0079] The Preparation of Polypeptide of the Present Invention

[0080] The polypeptide of the present invention can be prepared bymethods known in the art, such as, chemical synthetic methods andrecombinant DNA technology. Preferably, the preparation method for thepolypeptide of the present invention can be through recombinant DNAexpression The methods for preparing recombinant protein are well-knownin the art, which is not necessary described in detail herewith; inaddition, reference could be made to the methods in examples. As to thecell useful for producing the recombinant protein, mention should bemade to bacterial cell (P. O. Olins and S. C. Lee, 1993, Curr. Opi,Biotechnology, 4:526-525), yeast cell (R. G. buckholz, 1993, Curr. Opi.Biotechnology, 4:538-542), animal cell, especially mammals cell culture(C. P. Edwards and A. Aruffo, 1993, Curr. Opi. Biotechnology,4:558-563), and insect cell. With respect to the method for insect cell,reference should be made to, e.g., baculovirus (V. K Luckow, 1993, Curr.Opi. Biotechnology, 4:564-572). In this regard, the present inventionfurther provides a recombinant expression vector, which comprising thenucleotide sequence encoding the above-mentioned polypeptide. Thepresent invention still further provides a host cell transformed withthe above recombinant expression vector, which is able to expressionsaid polypeptides encoded by the nucleotide sequence contained therein.

[0081] In one embodiment of the present invention, E.coli is used toindividually express the polypeptide of the present invention asfollows: polypeptide 241, polypeptide 232, polypeptide 222, polypeptide201, polypeptide 235N, polypeptide 225N, polypeptide 209N, polypeptide208N, polypeptide NE2I, polypeptide 217D, polypeptide 205, polypeptide189, polypeptide 188, and polypeptide a) NE2; b)193C.

[0082] The ratio between monomer and dimer of said polypeptide, theformation of polymeric polypeptide and individual radium thereof aredetermined (see, Example 6 for detail). As the result shows that theexpressed product is prone to refold. The capability for self-formingstable polymer in solution after refolding makes the polypeptide of thepresent invention particularly suitable to be used as vaccine, for theprophylaxis and/or treatment of HEV. In one of the embodiment in thepresent invention, the polymer including dimmer, trainer, and tetramerhas been detected in one test. Limited by the methodology up to date,there is possibility that the deduced trimmer is in fact composed of themixture of dimmer and tetramer in a suitable ratio. It is alsocontemplated that the polypeptide of the present invention is able toform even larger polypeptide due to the improvement on methodology.Deducing from published reference (Jameel, et al., 1996, J. Virology,70:207-216; Li, et al., 1999, Virology, 265:35-45), there is highpossibility that natural HEV is composed of 90 sub-particles, whereineach sub-particles is dimmer of ORF2 polypeptide. Therefore, it isreasonably contemplated that the polypeptide of the present invention isable to form a polymer of up to 180-meric polypeptide, or even largerpolymers, with the development on the understanding of virus structure.

[0083] In another preferred embodiment in the present invention,although polypeptide 201 is expressed by E.coli in form of inclusionbody with high yield, the present inventor surprisingly finds that saidinclusion body is able to spontaneously self-renatured in the PBS bufferof pH7.45, which avoids the time-costing and tedious conventionaldenature/renature process that substantively reduce recovering rate,including the steps of adding guanidine hydrochloride and thenundergoing multi-steps of dialysis. In addition, since other non-desiredprotein inclusion body simultaneously expressed by E. coli is unable tospontaneously self-renature, the protein of interest in the presentinvention can be substantively purified simply by centrifugation andrecovering supernatant.

[0084] The Chimeric Protein Consisting of Polypeptide of the PresentInvention and Conserved Fragment of Hemagglutintin Antigen fromInfluenza Virus

[0085] In another aspect of the present invention, chimeric proteinconsisting of any one of the above-mentioned polypeptides and conservedfragment of hemagglutintin antigen from influenza virus is alsoprovided. Hemagglutintin antigen (hereinafter designed as HA) is one thetwo surface antigens of influenza virus, and is the most importedantigen used in specific detection for antibody against influenza virusin the serum of subject. It is known that antibody raised by vaccinatinganimal with HA can effectively prevent the receipt from re-infection ofinfluenza virus. Therefore, it is reasonable to believe that antibodyagainst HA is presented in most of population. According to previousreport (McEwen J. et al., Vaccine, 1992;10 (6):405-11), epitope 91-108aa is the conserved amino sequence in HA gene among all the H3 strainsof influenza virus type A. In one preferred embodiment of the presentinvention, firstly, chimeric expression in prokaryotic expressionsystem, especially in E. coli is established by flexibly linking HA gene(91-108aa) to the polypeptide fragment of HEV ORF2 gene of the presentinvention that is highly immunogenic, such as Gly-Gly-Ser by geneticengineering. Then boost with HA antibody raised by previously infectionof influenza virus, so as to generate high-titer protective anti-HEVantibody. In this way, a HEV vaccine is obtained that is super to thevaccine containing ORF2 fragment of polypeptide of the present inventionalone.

[0086] In light of the teachings in the present invention, epitopesuseful to the vaccine composition of the present invention can beselected from other conserved fragment in HA gene by skilled person inthe art. As to the specific flexible linker linking specific epitope ofHA and polypeptide of the present invention, it can be consisted ofsuitable peptide fragment or analog thereof, provided that it facilitatelinkage between polypeptide of the present invention and selectedfragment of HA and that does not substantively affect the use ofpolypeptide of the present invention for prophylaxis/treatment HEVinfection in mammals. It should be understand, selection of linker forlinking polypeptide of the present invention and HA mainly depends onthe specific property of the selected polypeptide of the presentinvention. For example, different linkers might be selected according toselected polypeptide of the present invention to be linked with HA.Preferably, the conserved fragment of HA used in the present inventionis a fragment from amino acid residue 91-108.

[0087] Linker for the linkage of polypeptide of the present invention asimmunogen and selected fragment of HA can be synthesized preferably byconventional synthesis technique, such as chemical synthesis technique.In addition, any peptide can be synthesized by skilled person in the artin accordance with standard chemical method, such as t-BOC method byautomatic peptide synthesisor (see, e.g., L. A. Carpino, J. Am. Chem.Soc., 79:4427, 1957). However, peptide can also be produced bychemically hydrolysis of protein or other known methods.

[0088] Alternatively, the chimeric protein of polypeptide of the presentinvention with HA can be produced by host cell transformed with nucleicacid sequence of a DNA molecule, wherein said DNA molecule comprise asequence encoding fragment of HA and polypeptide of the presentinvention which is obtained by cloning in host microorganism or cellthrough conventional genetic engineering method, such as recombinant DNAtechnique. When it is generated by recombinant technique in thetransformed cell, the resulted chimeric protein can be purified andrecovered by routine method from culture medium, host cell or from theboth. Said chimeric protein produced by recombination method is isolatedso that the resulted peptide can be substantively separated from cellsubstance or culture medium during the recombinant production byrecombinant DNA techniques. In addition, coding sequence for theresulted peptide could also be prepared by synthesis, or by using virusRNA in accordance known method or the available plasmid containing cDNAthereof.

[0089] For use in the present method, the above-mentioned chimericprotein can be designed to generally known construct or other constructin order to increase the production thereof or facilitate purificationof the same. The suitable system and vectors is known and publicavailable, or commercial available for cloning and expression chimericpeptide in various microorganisms and cells, including such as E. coli,Bacillus, Streptomyces, Saccharomyces, mammals, yeast, insect cell andplant cell.

[0090] The chimeric protein produced by either recombination orsynthesis can be purified by routine purification method. The skilledperson in the art can easily determine a desired purity for thepolypeptide according to the use of interest.

[0091] Vaccine Composition

[0092] In another aspect of the present invention, it further provides avaccine composition for prophylaxis and/or treatment of hepatitis Evirus infection in mammals, which comprises at least one of polypeptidesof the present invention or any combination thereof, and optionally,pharmaceutically acceptable vehicles and/or adjuvant.

[0093] In still another aspect of the present invention, it furtherprovides a vaccine composition for prophylaxis and/or treatment ofhepatitis E virus infection in mammals, which comprises chimeric proteincontaining a polypeptide of the present invention and a conservedfragment from hemagglutin antigen of influenza virus, and optionally,pharmaceutically acceptable vehicles and/or adjuvant.

[0094] In still another aspect of the present invention, use of abovevaccine compositions for vaccinating mammals to prevent from hepatitis Evirus infection is provided.

[0095] In present invention, mammals to be inoculated or treatedincludes but not limits to human being and other primates, such asbaboon, ape, monkey, etc.; economic animals, such as, bovine, caprine,swine, rabbit, murine, as well as pets, such as feline, canine, etc.Said vaccine composition contains treatment and/or prophylaxis effectiveamount of at least one of polypeptide of the present invention, whereinsaid effective amount is the amount that sufficient for effectivelytreat subject infected by HBV or prevent subject from HEV infectionafter administrating for a certain time.

[0096] The vaccine composition of present invention could be used eitheralone or as part of the formulation for medicament or prophylaxis, whichoptionally contains pharmaceutically acceptable vehicles, includingrelease-controlling agent. Said vehicles might further includepharmaceutically acceptable vectors or diluents suitable for theadministration for treatment and/or prophylaxis of HEV infection.Suitable pharmaceutically acceptable vectors refer to those biologicallyinert and/or non-toxic. Various vectors known in the art can be selectedaccording to desired use. Typically, said vector can be selected frombut not limit to the group consisting of: sterile saline, lactose,sucrose, calcium orthophosphate, gelatin, dextrin, agar, alum, aluminumoxide, aluminum hydroxide, peanut oil, olive oil, sesame oil, and water.Additionally, vector or diluents can further include controlled releasedsubstance, such as glyceryl monostearate/glyceryl distearate, eitheralone or in combination with paraffin. In addition, conventionalcontrolled release polymer formulation, including soluble glass can alsobe used.

[0097] Still further, when desired, the vaccine composition of thepresent invention comprising at least one polypeptide of presentinvention or any combination thereof can further comprise othertreatment/prophylaxis agent. For example, said composition couldcomprise a “cocktail mixture” of various agent that is useful in thetreatment or prophylaxis for HEV infection. Such cocktail mixture couldfurther include other agents, such as interferon, nucleotide analogsand/or N-acetyl-cysteine.

[0098] Optionally, the vaccine composition of the present inventioncomprising at least one polypeptide of present invention might furthercomprise immune system modifiers, such as, adjuvants or cytokines usefulfor further induction of antibody and T cell response in subject. Saidmodifier includes conventionally alum-based adjuvants, muramyldipeptides, preservatives, chemical stabilizers or other antigenicprotein. Generally, stabilizers, adjuvants or preservatives and the likeare optimized of dertrin the best formulation for efficacy in thedesired application. Suitable preservatives may include chlorylbutynol,potassium sorbate, sorbaic acid, sulphur dioxide, propyl galade,parabens, glycerine, and phenol.

[0099] Method for Prophylaxis and/or Treatment of HEV Infection inMammals using Vaccine Composition of the Present Invention

[0100] In another aspect, the present invention provides a method forprophylaxis and/or treatment of hepatitis E virus infection in mammals,which comprises administrating to the subject with a prophylaxis and/ortreatment effective amount of polypeptide (s) of the present inventionor chimeric protein (s) consisting of at least one of polypeptides ofpresent invention and conserved fragment of hemagglutin of influenzavirus. In particularly, said method comprises the step of administratingto subject with the vaccine composition of the present invention.Preferably, the conserved fragment selected by the present invention isamino acid fragment from 91-108 amino acid residues.

[0101] Suitable amounts of these compositions may be determined based onthe level of response desired. In general, compositions comprising thepolypeptide of present invention may contain between about 5 ug andabout 200 ug of the particles. Such compositions may be administrated asone or a series of inoculations, for example, three inoculations atintervals of two to six months. Suitable dosage may also be determinedby judgment of the treating physician, taking into account factors suchas the patient's health status, weight or age, as well as theconventional dosage of a component immunogen, when administered as amonotherapy Upon improvement of a patient's condition or likelihood ofincrease exposure to a given pathogen, a maintenance dose of acomposition comprising polypeptide of present invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced to a level at which the desiredeffect is retained. At that point, treatment should cease. Individualsmay, however, require intermittent treatment on a long-term basis uponrecurrence of a given unwanted condition.

[0102] Compositions comprising polypeptide of present invention may beadministered by any suitable route, such as, for example, parenteraladministration, particularly intramuscular or subcutaneous, as well asoral administration. Other routes, may also be used, such as pulmonary,nasal, aural, anal, dermal, ocular, intravenous, intraarterial,intraperitoneal, mucosal, sublingual, subcutaneous and intracranial.

[0103] Preparation of vaccine composition of present invention may becarried out to formulate injectable compositions or vaccine, either asliquid solutions or suspensions. Solid forms suitable for solution orsuspension in liquid prior to injection may also be prepared.Preparations also may, in certain embodiments, be emulsified orencapsulated in liposomes, or in soluble glasses, for controlledreleased and /or prolonged delivery. Alternatively, preparations may bein aerosol or spray form. They may also be included in trans-dermalpatches. The active ingredient may be mixed with any number ofexcipients which are pharmaceutically acceptable and compatible with theactive ingredient or ingredients. The excipients include, for example,Freund's incomplete adjuvant, bacterial lipopolysaccharides, ionexchanger, alumina, aluminum stearate, muramyl dipeptide, lecithin,buffer substance, cellulose-base substances and polyethylene glycol.

[0104] Diagnostic Kit and Method for Detecting Antibody IgG, IgM orTotal Antibody Against HEV in Biological Sample

[0105] The polypeptide of the present invention can be used fordetection of presence of antibody IgG, IgM or total antibody against HEVin biological sample, which is characterized in high sensitivity andhigh specificity, compared to exited detection kit or detection method.Therefore, the present invention provides a method for determine thepresence of HEV infection in a biological sample, which comprises thestep of contacting sample to be detected with detection effective amountof polypeptide of the present invention under a condition that suitablefor antibody/antigen interaction.

[0106] The biological sample to be detected in the present inventionderived from, includes but not limits to, human being and otherprimates, such as baboon, ape, monkey, etc.; economic animals, such as,bovine, caprine, swine, rabbit, murine, as well as pets, such as feline,canine, etc.

[0107] In another aspect of the present invention, the diagnostic kitfor determination of antibody IgG against hepatitis E virus in thebiological sample is provided, which comprises at least one of thepolypeptides of the present invention, if desired, said polypeptide ispre-coated on the surface of a suitable support; and further comprisescommercial available or routinely generated, detectable labeled antibodyanti-IgG that is directed against IgG from biological sample to bedetected, and detection agent corresponding to said detectable label;and if desired, further comprises a suitable buffer system.

[0108] In one embodiment of present invention, said biological sample tobe detected is derived from human being, wherein antibody is anti-humanIgG antibody. More specifically, the diagnostic kit for IgG antibody ofthe present invention further includes a polypeptide having immunogenicepitope in HEV ORF3 or an immunogenic fragment thereof, wherein saidimmunogenic epitope in HEV ORF3 or an immunogenic fragment thereof isoptionally covalently bound to the polypeptide of present invention.

[0109] For the situation that said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof is optionally covalently bound to thepolypeptide of present invention, the chimeric polypeptide is preferablyproduced by genetic recombination method. Chemical method can also byused to covalently bind said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof to the aforementioned polypeptide.

[0110] In another aspect of the present invention, a diagnostic kit fordetermination of antibody IgM against hepatitis E virus in thebiological sample is provided, which comprises commercially available orroutinely generate detectable labeled antibody anti-IgM as captureantibody that is directed against IgM from biological sample to bedetected, if desired, said capture antibody is pre-coated on the surfaceof a suitable support; and further comprises detectable labeled at leastone of the polypeptides of the present invention, and detection agentcorresponding to said detectable label; if desired, further comprises asuitable buffer system.

[0111] In one embodiment of present invention, said biological sample tobe detected is derived from human being, wherein antibody is anti-humanIgM antibody. More specifically, the diagnostic kit for IgM antibody ofthe present invention further includes a polypeptide having immunogenicepitope in HEV ORF3 or an immunogenic fragment thereof, wherein saidimmunogenic epitope in HEV ORF3 or an immunogenic fragment thereof isoptionally covalently bound to the polypeptide of present invention.

[0112] For the situation that said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof is optionally covalently bound to thepolypeptide of present invention, the chimeric polypeptide is preferablyproduced by genetic recombination method. Chemical method can also byused to covalently bind said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof to the aforementioned polypeptide.

[0113] In another aspect of the present invention, a diagnostic kit fordetermination of antibody IgM against hepatitis E virus in thebiological sample is provided, which comprises commercially available orroutinely generated, detectable labeled antibody anti-IgM as captureantibody that is directed against IgM from biological sample to bedetected, if desired, said capture antibody is pre-coated on the surfaceof a suitable support; and further comprises detectable labeled at leastone of the polypeptides of the present invention, and detection agentcorresponding to said detectable label; if desired, further comprises asuitable buffer system.

[0114] In one embodiment of present invention, said biological sample tobe detected is derived from human being, wherein antibody is anti-humanIgM antibody. More specifically, the diagnostic kit for IgM antibody ofthe present invention further includes a polypeptide having immunogenicepitope in IEV ORF3 or an immunogenic fragment thereof, wherein saidimmunogenic epitope in HEV ORF3 or an immunogenic fragment thereof isoptionally covalently bound to the polypeptide of present invention.

[0115] For the situation that said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof is optionally covalently bound to thepolypeptide of present invention, the chimeric polypeptide is preferablyproduced by genetic recombination method. Chemical method can also byused to covalently bind said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof to the aforementioned polypeptide.

[0116] In still another aspect of present invention, a diagnostic kitfor determination of total antibodies against hepatitis E virus in thebiological sample is also provided, which comprises at least one of thepolypeptides of present invention, if desired, said polypeptide ispre-coated on the surface of a suitable support; and further comprisesdetectable labeled at least one of polypeptides according to claim 1,and detection agent corresponding to said detectable label; wherein saidpolypeptide selected from polypeptides according to claim 1 forpre-coating the surface of a support and the detectable labeledpolypeptide selected from polypeptides according to claim 1 could be thesame polypeptide, or different one.

[0117] More specifically, the diagnostic kit for total antibodies of thepresent invention further includes a polypeptide having immunogenicepitope in HEV ORF3 or an immunogenic fragment thereof, wherein saidimmunogenic epitope in HEV ORF3 or an immunogenic fragment thereof isoptionally covalently bound to the polypeptide of present invention.

[0118] For the situation that said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof is optionally covalently bound to thepolypeptide of present invention, the chimeric polypeptide is preferablyproduced by genetic recombination methods. Chemical method can also byused to covalently bind said immunogenic epitope in HEV ORF3 or animmunogenic fragment thereof to the aforementioned polypeptide.

[0119] It is well known in the art that in the aforementioned diagnostickit, anti-human IgG or anti-human-IgM which can be generated by variouscommercial available or routinely generated method taking advantage ofvarious animals are used Alternatively, anti-IgG or anti-IgM against thespecific animals from which biological sample to be detected derived areused which can be generated by taking advantage of relating animals. Forthe purpose of preparing antibody, selected animal includes but notlimit to goat, sheep, rat, mouse, rabbit, guinea pig, swine, etc. Saiddetectable label for labeling can be used alone or in combination withother composition or compound for providing detectable signal tovisualize the presence of substance of interest in sample. Saiddetectable label can be those known and easily available materials inthe art of detection field, including but not limited to enzyme marker,fluorescent marker, radioactive marker, etc. Thus, the present inventionis not limited to specific selection of detection label, andcontemplates that it includes all those detection method known in theart. For the purpose of convenience, said detection agent can beprovided in the form of kit.

[0120] Optionally, said kit further includes micro-titer platepre-coated with polypeptide of present invention, various suitablyformulated diluent and/or buffer, labeled substance or othersignal-generating agent for detection of specifically boundantigen/antibody complex, such as, enzymatic substrate, co-factor andchromophore, Other components therein could be easily selected byskilled person in the art.

[0121] Additionally, a method for detecting antibody IgG againsthepatitis E virus in biological samples is provided, which comprises thestep of: immobilizing at least one of the polypeptides of presentinvention on the surface of a support; then washing with a suitablebuffer; contacting it with sample to be detected under the conditionssuitable for the interaction of antigen and antibody; washing again witha suitable buffer; and then incubating with commercial available orroutinely generated, detectable labeled anti-IgG antibody a certain timesufficient for antigen/antibody interaction, wherein said anti-IgG isagainst the animal from which the biological sample to be detectedderived; after that, detecting the antigen/antibody complex on thesurface of a support by using detect agent corresponding to saiddetectable label, calculating the amount of antibody IgG in the sample

[0122] In one embodiment of present invention, said biological sample tobe detected is derived from human being, said antibody as used isanti-human IgG.

[0123] In another embodiment of present invention, polypeptide NE2I asantigen is pre-coated to the surface of a predetermined support. Inanother embodiment of present invention, polypeptide 247 coupled withepitope in HEV ORF3 is used as antigen to be pre-coated with specificsurface of support.

[0124] In another aspect of present invention, a method for detectingantibody IgM against hepatitis B virus in biological samples isprovided, which comprises the step of: immobilizing commercial availableor routinely generated antibody ant-IgM on the surface of a support,wherein said anti-IgM is against the animal from which biological sampleto be detected derived; washing suitably; contacting it with sample tobe detected, preferably serum, under the conditions suitable for theinteraction of antigen and antibody; washing again with a suitablebuffer; and then incubating with detectable labeled at least one ofpolypeptide of present invention for a time sufficient for theinteraction between antigen and antibody, after that, detecting theantigen/antibody complex on the surface of a support by using detectagent corresponding to said detectable label, calculating the amount ofantibody IgM in the sample.

[0125] In one embodiment of present invention, said biological sample tobe detected is derived from human being, said antibody as used isanti-human IgM.

[0126] In another embodiment of present invention, said IgM antibody insample is detected by polypeptide 225N coupled with horseradishperoxidase. In still another embodiment of present invention,polypeptide 247 coupled with epitope of HEV IRF3 is further coupled withhorseradish peroxidase to detect IgM contained in sample.

[0127] Additionally, a method for detecting total antibodies againsthepatitis E virus in biological samples is also provided, whichcomprises the step of: immobilizing at least one of the polypeptides ofpresent invention on the surface of a support; washing with a suitablebuffer; contacting it with biological sample to be detected under theconditions suitable for the interaction of antigen and antibody;optionally, washing again with a suitable buffer; incubating withdetectable labeled one of present polypeptide for a time sufficient forthe interaction between antigen and antibody; and detectingantigen/antibody complex on the surface of a support by using antigen ofhepatitis E virus with a detectable label and corresponding detectagent, calculating the amount of total antibodies in the sample.

[0128] In one embodiment of present invention, NE2I is pre-coated on thesurface of support, and total antibodies in the sample is detected bypolypeptide 225 previously coupled with horseradish peroxidase. Inanother embodiment of present invention, NE2I coupled with epitope fromHEV ORF3 is precoated on the surface of support, and total antibodies inthe sample is detected by horseradish peroxidase bound polypeptide 225which is previously coupled with epitope from HEV ORF3.

[0129] The present invention is further illustrated in details withreference to the following description of drawings and examples, whichshould not in any way be interpreted as the limitation to the protectionscope of the present invention.

EXAMPLES

[0130] Unless specifically indicated, experiment methods of molecularbiology and immunoassays of the present invention are all followingthose basically described in Molecular Cloning: a Laboratory Manual, 2ndEdition, Joseph Sanbrook, David W. Russell, by Cold Spring HarborLaboratory Press and Short Protocols in Molecular Biology,3rd Edition,John Wiley & Sons,Inc.,1995. The use of restriction endonucleasesfollows the protocols provided by the producer.

Example 1

[0131] Preparation of the Genes Encoding the Polypeptides of the PresentInvention and Construction of Expression Vector Containing the Same

[0132] Preparation of the Fraction of HEV ORF2 as Template.

[0133] To prepare gene of interest, polymerase chain reaction (PCR) isused with a full-length HEV gene cloned from HEV-infected patient in XinJiang provine, China as template (Aye, T. T., Uchida etc., Nucleic AcidsResearch, 20(13),3512(1992); GeneBank accession number D11092), togetherwith two primers, ORF2 FP:5′-atgcgccctcggcca-3′ as upper primer and ORF2RP: 5′-aaataaactataactcccga-3′as lower primer. PCR reaction is carriedout in PCR thermal cycler (BIOMETRA t3) under following condition: 94°C. 5 min; then 25 circles of: 94° C. 50 sec, 57° C. 50 sec and 72° C.2.5 min; ended by 72° C. 10 min. A DNA fragment about 2 kb is obtained,which is from HEV ORF 2 as the template for preparation of polypeptideof the present invention. The above-mentioned PCR product is furtherlinked into commercial available vector pMD18-T (TAKARA CO.) and thendigested with BamH I/Hind III, so as to identify the positive cloneinserted with ORF2 gene. Using M13 (+)/(−) as primer, the resultant issequenced and thereby identify the two DNA fragment of HEV ORF 2 whichis used as the template for preparing polypeptide of present invention,one of them is a conservative sequence (Template 1, SEQ ID NO; 5),theother is a mutant sequence (Template 2, SEQ ID NO: 6).

[0134] By sequence alignment and analysis of ORF, it is found that themutant sequence of HEV ORF2 (SEQ ID NO: 6) as template for preparing thepolypeptide of present invention has a base A deleted, compared toconservative sequence (SEQ ID NO: 5), which resulted in shift mutationthat amino acid residues 604-605 in ORF2 are mutated from His-Ser-Val toPro-Pro-Arg, and the translation for said polypeptide is thereby stoppedby stop code tag formed by such mutation.

[0135] By way of example, the potypeptide 201 of present invention isused hereafter to illustrate the preparation of nucleotide encodingpolypeptide 201 and the expression vector comprising the same.

[0136] The Preparation of Polynucleodide Encoding Polypeptide 201 ofPresent Invention and the Expression Vector Containing the Same.

[0137] The gene is synthesized using polymerase chain reaction (PCR),wherein the above-obtained sequence SEQ ID NO: 5 is used as template,together with forward primers,201FP:5′-ggatcecatatggttattcaggattatgac-3′ (see, table 1), in whichBamHI sites, NdeI sites (CAT ATG), and ATG as translation start codon inE. coli system are introduced; and 201RP:5′-otcgagasataaactataactcccga-3′ (see, Table 1) as reversed primer, inwhich stop codon and EcoR I site are introduced. The PCR is carried outin thermo cycler as follows: heat-denatured for 5 min at 94° C.,thenamplified for 30 circles: 50 sec at 94° C., 40 sec at 57° C., and 40 secat 72° C., finally 10 min at 72° C. The resulted ˜600 bp PCR product isidentified as the nucleotide sequence encoding polypeptide 201 ofpresent invention.

[0138] The construction of expression vector pTO-T7 for expressing thepolypeptide of present invention is following the method in referencedocument of LUO Wen-Xin, et al., Chinese Journal ofBiotechnology,2000,16:53-57. Said method includes steps of: cloningaforementioned PCR product into commercial available pMD18-T vector(TAKARA company), and digesting with BamHI/HindIII to identify andobtain the positive subclone inserted with nucleotide encodedpolypeptide 201; further digesting said positive subclone with NdeI andEcoRI to obtain nucleotide sequence comprising gene of polypeptide 201,and then cloning into NdeI/EcoRI-digested pTO-T7. The positive clonepTO-T7-ORF2-201 that embodying encoding sequence of the polypeptide 201is identified by digestion with NdeI/EcoRI. The strategy forconstructing the expression vector for polypeptide of ORF 201 isillustrated in FIG. 1.

[0139] Similarly, other polypeptides of present invention whose carboxylterminus is other than Pro-Pro-Arg can be acquired according to theabove method by using sequence of SEQ ID NO: 6 as template, and usingprimers listed in Table that is specifically designed against individualpolypeptide of interest.

[0140] The nucleotide encoding polypeptides of present invention whosecarboxyl terminus is Pro-Pro-Axg and expression vectors containing thesame.

[0141] The polypeptides of present invention whose carboxyl terminus isPo-Pro-Arg is expressed by transforming E. coli ERR 2566 with expressionvector obtained according to the above-mentioned method for expressionvector of ORF2-201. Specifically, taken above-mentioned HEV ORF2 mutantsequence SEQ ID NO: 6 as template, using individual forward/reverseprimer specifically designed against individual polypeptide of presentinvention (see, Table 1), corresponding expression vector is obtained byPCR under similar the condition for generating expression vector ofpolypeptide 201. In this way, a series of polypeptide of presentinvention with good immunogenicity and immunoreactivity is obtained,wherein the resulted polypeptide has Met added at its N-terminus, andhas amino acid sequence -Pro-Pro-Arg added in direction from 5′-3′ at 3′end of amino acid 603. Pro, at its carboxyl terminus. TABLE 1 PCRamplification template for the preparation of nucleotide encodingpolypeptide of present invention and corresponding forward/reversedprimers position intemplate polypeptide HEV ORF2 No. forward primer (FP)and reversed primer (RP) NE2 394-603ppr* 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ 2 HERP:5′-ctcgagaaataaactataactcccga-3′ 217C 390-603ppr  2 217FP:5′-ggatcccatatgtcggctggtggccag-3′ HERP: 5′-ctcgagaaataaactataactcccga-3′193C 414-603ppr  2 E220F: 5′-ggatcccatatgacatctgtagagaatgctca-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 178C 429-603ppr  E235F:5′-ggatcccatatgcatgacatcgacctcg-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 168C 439-603ppr  E46F:5′-ggatcccatatggttattcaggattatgac-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 158C 449-603ppr  2 E56F:5′-ggatcccatatgcaggaccgaccgac-3′ HERP: 5′-ctcgagaaataaactataactcccga-3′148C 459-603ppr  E66F: 5′-ggatcccatatgtcgcgcccttttt-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 138C 469-603ppr  2 138CF:5′-ggatcccatatggacgtgctttggctttctc-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ NE2D 394-603 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ E2RD:5′-gaattcttagggggctaaaacagc-3′ 217D 390-603 2 217FP:5′-ggatcccatatgtcggctggtggccag-3′ E2RD: 5′-gaattcttagggggctaaaacagc-3′193D 414-603 2 E220F: 5′-ggatcccatatgacatctgtagagaatgetca-3′ E2RD:5′-gaattcttagggggctaaaacagc-3′ 178D 429-603 2 E235F:5′-ggatcccatatgcatgacatcgacctcg-3′ E2RD: 5′-gaattcttagggggctaaaacagc-3′NE2I 394-606 2 HEFP: 5′-ggatccatatgcagctgttctactctcgtc-3′ E2RI:5′-gaattctatgcggaatggggggctaaaacag-3′ 217I 390-606 2 217FP:5′-ggatcccatatgtcggctggtggccag-3′ E2RI:5′-gaattcttatgcggaatggggggctaaaacag-3′ 193I 414-606 2 E220F:5′-ggatcccatatgacatctgtagagaatgctca-3′ E2RI:5′-gaattcttatgcggaatggggggctaaaacag-3′ 178I 429-606 2 E235F:5′-ggatcccatatgcatgacatcgacctcg-3′ E2RI:5′-gaattcttatgcggaatggggggctaaaacag-3′ 266N 394-660 1 HEFP:5′-ggatccatatgcagctgttotactctcgtc-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 235N 394-628 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ 235NR:5′-gaattcttacgggcagaagtcatcg-3′ 225N 394-618 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ 225RP:5′-gaattcttaggcagggtagtccatgg-3′ 209N 394-602 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ 209RP:5′-gaattcttaggctaaaacagcaacc-3′ 208N 394-601 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ 208RP:5′-gaattcttataaaaoagcaaccgc-3′ 207N 394-600 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ 207RP: 5′-gaattcttaaacagcaaccgcg-3′203N 394-596 2 HEFP: 5′-ggatccatatgcagctgttctactctcgtc-3′ E203R:5′-gaattcttaggaaatagagacgggac-3′ 193N 394-586 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ E220R:5′-ctcgagttaagtggtgtaagtggaaatag-3′ 176N 394-569 2 HEFP:5′-ggatccatatgcagctgttctactctcgtc-3′ E237R:5′-ctcgagttacagttggtcactagcagt-3′ 280  380-660 1 227FP:5′-ggatcccatatgctaggcggtctaccca-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 270  390-660 1 217FP:5′-ggatcccatatgtcggctggtggccag-3′ HERP: 5′-ctcgagaaataaactataactcccga-3′260  400-660 1 207FP: 5′-ggatcccatatgcccgtogtctcagc-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 247  414-660 1 E220F:5′-ggatcccatatgacatctgtagagaatgctca-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 232  429-660 1 E235F:5′-ggatcccatatgcatgacatcgacctcg-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 222  439-660 1 E46F:5′-ggatcccatatggttattcaggattatgac-3′ HERP:5′-ctcgagaaataaactataactcccga-3′ 205  414-618 2 E220F:5′-ggatccoatatgacatctgtagagaatgctca-3′ 225RP:5′-gaattcttaggcagggtagtccatgg-3′ 201  459-660 1 E46F:5′-ggatcccatatgggtattcaggattatgac-3′ HERP:5′-ctcgagaaaaaactataactcccga-3′ 191  469-660 1 138CF:5′-ggatcccatatggacgtgctttggctttctc-3′ HERP:5′-ctcgagaaataactataactcccga-3′ 189  414-602 2 E220F:5′-ggatcccatatgacatctgtagagaatgctca-3′ 209RP:5′-gaattcttaggctaaaacagcaacc-3′ 188  414-601 2 E220F:5′-ggatcccatatgavatctgtagagaatgctca-3′ 208RP:5′-gaattcttataaaacagcaaccgc-3′ 183  414-596 2 E220F:5′-ggatcccatatgacatctgtagagaatgctca-3′ E203R:5′-gaattcttaggaaatagagacgggac-3′ 173  414-586 2 E220F:5′-ggatcccatatgacatctgtagagaatgctca-3′ E220R:5′-ctcgagttaagtggtgtaagtggaaatag-3′ 170  459-628 2 E46F:5′-ggatcccatatggttattcaggattatgac-3′ 235NR:5′-gaattcttacgggcagaagtcatcg-3′ C160 469-628 2 138CF:5′-ggatcccatatggacgtgctttggctttctc-3′ 235NR: 5′-gaattcttacgggcagaagtcatcg-3′ N160 459-618 2 E46F:5′-ggatcccatatggttattcaggattatgac-3′ 225RP:5′-gaattcttaggcagggtagtccatgg-3′ 150  469-618 2 138CF:5′-ggatcccatatggacgtgctttggctttctc-3′ 225RP: 5′-gaattcttaggcagggtagtccatgg-3′ 144  459-602 2 E46F:5′-ggatcccatatggttattcaggattatgac-3′ 209RP:5′-gaattcttaggctaaaacagcaacc-3′ 142  459-600 2 E46F:5′-ggatcccatatggttattcaggattatgac-3′ 207RP: 5′-gaattcttaaacagcaaccgcg-3′134  469-602 2 138CF: 5′-ggatcccatatggacgtgctttggctttctc-3′ 209RP:5′-gaattcttaggctaaaacagcaacc-3′

Example 2

[0142] Expression of Polypeptide 201

[0143] 1 uL plasmid pTO-T7-ORF2-201 (0.15 mg/mL) was added into 40 uLcompetent cells of E. coli ERR2566 (generated under calcium chloridemethod) for transformation. Then the mixture was scrawled onto akanamycin-LB plate, and the plate was incubated at 37° C. for 10˜12hours till the present of individual clones. The individual clones werepicked and further inoculated in 4 mL LB culture medium in tubes, 220rpm shaking at 37° C. for 10 hours, until the OD_(550 nm) value of theculture is about 1.5. Then 1 mL culture medium was stored at 4° C. forlater use, and 2 uL 0.5M IPTG was added into the rest 3 ml of culturemedium (final content is 0.3 mM). The culture medium contain IPTG waskeep on incubating at 37° C. for 4 hours with 220 rpm shaking forinducing the expression of polypeptide of interest. 1.5 mL inducedculture medium was centrifuged at 12000 g for 30 seconds. Theprecipitated cells were resuspended in 100 uL protein loading buffer (50mM Tris Cl pH6.8, 100 mM DTT, 2% SDS, 0.1% Bromophenol Blue, 10%Glycerol), and further boiled for 10 minutes, then centrifuged at 12000g for 10 minutes. 10 ul supernatant was load onto 12% SDS-PAGE foranalysis of the expression of polypeptide 201. The clone which expressin highest yield was used for future fermentation.

[0144] 200 uL seek culture medium was added into 500 mL LB culturemedium containing in 1L Erlenmeyer flask. After incubating at 37° C.with 190 rpm shaking for about 11 hours till the OD_(500 nm) value ofthe culture reached 2.0, 300 uL 0.5M IPTG was added to the final contentis 0.3 mM. And the mixture was further incubated for 4 hours underaforementioned condition. 1.5 mL induced culture was centrifuged at12000 g for 30 seconds. The cells were resuspended in 100 uL proteinloading buffer, and boiled for 10 minutes, then centrifuged at 12000 gfor 10 minutes. 10 ul supernatant was load onto 12% SDS-PAGE foranalysis of expression of polypeptide 201. The result of the analysis ofthe SDS-PAGE (staining with Coomassie Brilliant blue R250) showed theexpression of polypeptide 201 is about 35% of total expressed cellproteins as UVI gel imaging instrument shown (UVitech, model DDT-08).

Example 3

[0145] Purification of Polypeptide 201 Inclusion Body Expressed in E.coli

[0146] The culture medium of E. coli containing recombinant polypeptide201 obtained in example 2 was centrifuged at 4000 rpm for 15 minutes,and each 500 mL culture's precipitate was re-suspended in 15 mL lysisbuffer (50 mM Tris Cl, 10 mM EDTA and 300 mM NaCl in dH₂O, pH7.2). Thecells were sonicated in ultrasonic instrument (Uilbra-Ceil VCX500,SONICS&MATERIALS company, power 70%; 40 seconds on; 60 seconds off;sonicated for 20 minutes totally.). The sonicated mixture wascentrifuged 12000 rpm for 10 minutes at 4° C., and the pellet wasresuspended in buffer I solution (200 mM Tris Cl, pH8.5; 5 mM EDTA; 100mM NaCl) containing 2% Triton-X100, and the final volume is the same asthe original lysis. The mixture was shaking at 200 rpm for 30 minutes at37° C., then centrifuged at 10000 rpm for 10 minutes at 4° C. The pelletwas resuspended in equal volume of buffer I, and the mixture wassonicated (40 seconds on; 60 seconds off; power 70%; sonicated for 3minutes totally). After that the mixture was centrifuged (10000 rpm) for10 minutes at 4° C. The pellet was resuspended in buffer I containing 2%Triton-X100 to the final volume is the same as before. After the mixturewas shake (200 rpm) for 30 minutes at 37° C., it was centrifuged (10000rpm) for 10 minutes at 4° C. The pellet was resuspended in equal volumeof buffer I, shaking (200 rpm) for 30 minutes at 37° C. Then the mixturewas centrifuged 10000 rpm for 10 minutes at 4° C. The pellet wasresuspended in buffer I which containing 2M Urea to the final volume isthe same as original mixture. After shaking 200 rpm for 30 minutes at37° C., the mixture was centrifuged 10000 rpm for 10 minutes at 4° C.This supernatant is marked with 20I-2M. The pellet was resuspended inbuffer I containing 4M Urea to the final volume is the same as before.After shaking (200 rpm) for 1 hour at 37° C., the mixture was stored at4° C. overnight, and then it was centrifuged 12000 rpm for 10 minutes at4° C. This supernatant is marked with 20I-4M. The purity of all theabove samples is analyzed by 12% SDS-PAGE, The results are shown in FIG.3.

Example 4

[0147] Renaturation of Recombinant Polypeptide 201

[0148] 100 ml of sample 201-4M prepared according to Example 3 wasloaded into 2 dialysis bags (36 DM, retentate MW: 8000-10000, UnitedCarbon Compound, U.S.A.) and dialyzed under stirring in a 1L beaker,with 900 ml of 1×PBS (20×PBS (1L) containing 73.344 g of Na2HPO4.12H2O,4 g of KH2PO4, 163,632 g of NaCl, 4.024 g of KCl and pH 7.45) at 25° C.over night (10 hours), and then white precipitates were observed in thedialysis bags. Refreshing the dialysate and going on the dialysis, andthen the dialysate was refreshed every 3 hours for 4 times. Inprinciple, the content of urea in the sample would be 4×10−6M when thedialysis is over. The dialyzed sample was centrifuged at 25° C., 12000rpm for 10 minutes; the supernatant was filtrated with 0.22 μm filtermembrane for further purification; the pellet resuspended in 4Murea/buffer I can be used in a new dialysis during which precipitateswould also appear, but the concentration of the obtained protein samplewould be lower than that of the first obtained sample.

Example 5

[0149] Purification of Recombinant Polypeptide 201 with Gel FiltrationHPLC

[0150] The renatured 201 sample prepared according to the methods ofExample 4 was further purified by HPLC as below:

[0151] Instrument: Beckman System Gold Nouveau 125NMP/166NMP HPLC,

[0152] Column: TSK GEL SW3000 21.5 mm×60 cm,

[0153] Elution, 1×PBS pH 7,45,

[0154] Flow Rate: 4 ml/min,

[0155] Detection: UV at 280 nm,

[0156] Sample: 2 ml of 4M NE2 (8 mg/ml),

[0157] Collection: automatic apex collection of window mode,

[0158] Collection time; 1 tube/20 seconds,

[0159] Collection delay: 6 seconds.

[0160] The result shows that the molecule filtering is very elective inthe chromatogram but that the apex component contains monomers anddimersas well as proteins distributed equably between them. Aftertreated in boiling water for 10 minutes, the sample protein was analyzedby SDS-PAGE with 12% acrylamide for the monomer purity of the objectprotein peak, which is up to more than 95%. This demonstrates that inaddition to self-aggregated, ORF2-201 monomer also aggregates with othersmall proteins and interactions occur also among the multimers whichwere eluted together during chromatogram,

Example 6

[0161] Characterization of the Recombinant Polypeptide Products of theInvention

[0162] The recombinant polypeptide of the invention was constructed andexpressed according to the methods of Examples 1-5. Furthermore, eachrecombinant peptide was washed and dialyzed according to the methods ofExamples 3-4. In table 2 the corresponding amino acid position of eachrecombinant peptide in hepatitis E virus, the renaturation property ofthe expressed recombinant products and the proportions of the monomersand dimers in their SDS-PAGE as well as the formation of the multimersare provided. TABLE 2 Corresponding amino acids position of eachrecombinant peptide in hepatitis E virus, the renaturation property ofthe expressed recombinant products and the proportions of the monomersand dimers in their SDS-PAGE as well as the formation of the multimers.name of percent of percent renaturable polypeptide sequence no. monomerof dimer Multimerization by dialysis NE2 SEQ ID NO: 2  10% 90% Yes Yes193C SEQ ID NO: 3  5% 95% Yes Yes 178C SEQ ID NO: 4 100%  0% No Yes 168CSEQ ID NO: 7 100%  0% No Yes 158C SEQ ID NO: 8  60% 40% No Yes 148C SEQID NO: 9 100%  0% No Yes 138C SEQ ID NO: 10 100%  0% No Yes SEQ ID NO: 1NE2I aa394˜aa606  5% 95% No Yes SEQ ID NO: 1 217I aa390˜aa606  85% 15%No Yes SEQ ID NO: 1 193I aa414˜aa606 100%  0% No Yes SEQ ID NO: 1 178Iaa429˜aa606  60% 40% No Yes SEQ ID NO: 1 NE2D aa394˜aa603  80% 20% NoYes SEQ ID NO: 1 217D aa390˜aa603  20% 80% No Yes SEQ ID NO: 1 193Daa414˜aa603 100%  0% No Yes SEQ ID NO: 1 178D aa429˜aa603 100%  0% NoYes SEQ ID NO: 1 235N aa394˜aa628  10% 90% No Yes 225N SEQ ID NO: 1  4%96% No Yes aa394˜aa618 SEQ ID NO: 1 209N aa394˜aa602  25% 75% No Yes SEQID NO: 1 208N aa394˜aa601 100%  0% No Yes SEQ ID NO: 1 207N aa394˜aa600100%  0% No No SEQ ID NO: 1 203N aa394˜aa596 100%  0% No No SEQ ID NO: 1193N aa394˜aa586 100%  0% No No SEQ ID NO: 1 176N aa394˜aa569 100%  0%No No SEQ ID NO: 1 247 aa414˜aa660  10% 90% No Yes SEQ ID NO: 1 232aa429˜aa660  10% 90% No Yes SEQ ID NO: 1 222 aa439˜aa660  10% 90% No YesSEQ ID NO: 1 205 aa374˜aa618  10% 90% No Yes SEQ ID NO: 1 201aa459˜aa660  1% 99% No Yes SEQ ID NO: 1 189 aa414˜aa602  2% 98% No YesSEQ ID NO: 1 188 aa414˜aa601  4% 94% No Yes SEQ ID NO: 1 183 aa414˜aa596100%  0% No No SEQ ID NO: 1 173 aa414˜aa586 100%  0% No No

[0163] As shown in Table 2, the polypeptides of the invention which isincluded in the amino acid sequence of SEQ ID NO: 1 of HEV ORF2 has theability to be well-renatured, which is intend to exhibits a dimensionalstructures close to natural HEV protein, when their carboxyl terminalslocate between aa 601 (Leu) and aa 660 of SEQ ID NO: 1. Specifically,the peptides 247, 232, 222, 201, 235N, 225N, 209N, NE2I, 217D, 205, 189,188, NE2 (SEQ ID NO: 2) and 193C (SEQ ID NO: 3) was found to haveexpression bonds in the position corresponding to monomer moleculeweight and 2 folds of monomer molecule weight while the amounts ofdimmers are apparently over those of monomers. NE2 and 193C was found tohave obvious bonds in positions of bigger molecule weights, whichsuggests that said peptides tend to multimerize spontaneously.

[0164] After renaturable recombinant peptides in Table 2, 193C, 201,208N, 209N, NE2, 222, 225N, 232 and 247, were further purified by Gelfiltration HPLC and centrifuged each for 10 min at 20000 g and filteredwith 0.1 μm Al₂O₃ filter membrane, the dynamic radiuses of thesepeptides was measured by dynamic light scattering instrument(DYNAPRO99-D-50 dynamic light scattering instrument, produced by PROTEINSOLUTIONS) and their assemble status were speculated in Table 3. Theobtained molecule radius of each recombinant peptide is apparentlybigger than the predicted radius of the monomer. According to theputative molecule weights, it can be concluded that those peptides format least dimmers in the solution, and most of them form higher ordermultimers, which are in conformity with their behavior in SDS-PAGE. Infact, the polypeptides of the invention prepared with the above methodscan form multimers of up to 180 or more monomers. It was furtherdemonstrated that the polypeptides of the invention have unexpectedproperty, i.e. above said recombinant peptides expressed by E. colisystem tends multimerize spontaneously in PBS solution free ofdenaturing reagents and this is advantageous for increasing itsimmunogenicity as vaccine. TABLE 3 Detention of the aggregate status ofrenaturable recombinant peptides of the invention by dynamic lightscattering instrument. theoretical molecule putative weight of measuredmolecule poly- monomer radius weight peptide (KD) (nm) (KD) Putativeaggregate states 193C 21.2 3.44 47 dimer (21.2 × 2 = 42.4) 201 22.1 3.0862.7 trimer (22.1 × 3 = 66.3) 208N 22.9 3.57 66.1 trimer (22.9 × 3 =68.7) 209N 23 4.10 91. tetramer (23 × 4 = 92) NE2 24.4 4.04 90 tetramer(24.4 × 4 = 97.6) 222 24.4 3.72 73 trimer (24.4 × 3 = 73.2) 225N 24.83.91 82 trimer (74.4) tetramer (99.2) co-existing 232 25.5 3.97 85trimer (25.5 × 3 = 76.5) 247 27.2 4.28 101 dimer (27.2 × 4 = 108.8) 266N29.3 4.41 108 tetramer (29.3 × 4 = 117.2)

Example 7

[0165] The Physiochemical Properties of Polypeptide

[0166] Renaturation from the Inclusion Body

[0167] The inclusion body of the recombinant polypeptide prepared asdescribed in examples 1-3 was denaturalized by 4M urea, then dialyzedwith over 100 volumes of PBS, as described in example 4. The dialysatewas centrifuged at 12,000 rpm for 10 min. The supernatant contains someor all of the recombinant polypeptides, thereby demonstrating that saidrecombinant polypeptides are capable of renaturation.

[0168] The Polymerization of the Recombinant Peptides

[0169] In the analysis of the supernatant using conventional SDS-PAGE,the bands corresponding respectively to the monomer, dimer and polymerwere identified. The specificity of said bands were further confirmed byconventional Western blotting, thereby demonstrating that recombinantpolypeptide 201 forms a polymer after renaturation(see FIG. 4).

[0170] The Determination of the Molecular Size of Polypeptide 201 byLight-Scanning Technique

[0171] According to Example 6, polypeptide 201 was centrifuged at 20,000rpm for 10 min after purification with HPLC, then filtered with 0.1 umMillipore membrane of alumina. The filtrate was measured by dynamiclight-scanning instrument (DYNAPOR99-D-50, PROTEIN SOLUTION Com. Ltd.U.S.A) at 824.0 nm. The Regulation algorithm was used for calculation,and its applicability was confirmed by many standard samples. The radiusof the molecule is calculated from the dynamic radius corresponding tothe % Intensity peak. The solvent was set as sample buffer PBS. Themeasured results shown in FIG. 5 indicated that the mean radius ofpolypeptide 201 in denaturant-free solution was 3.08 nm, with thecalculated MW 62.7 KD(corresponding to the trimer). It is known to thoseskilled in the art that said polypeptide of the invention could actuallyform polymers of 180 monomers or more.

Example 8

[0172] Preparation of Mouse Anti-NE2 Monoclonal Antibodies

[0173] Establishment of the Hybridoma Cell Line

[0174] For the primary immunization, each Balb/C female mouse (6-8 weeksold) was inoculated with 5 ug recombinant antigen NE2 emulsifiedFreund's incomplete adjuvant (the total volume is 50 uL). Fifteen dayslater, the mouse was intramuscularly immunized for the second time withthe same mount of NE2 emulsified in incomplete Freud's adjuvant 30 dayslater, the mouse was then boosted intravenously (via the tail vein) with5 ug antigens without the adjuvant. The mice were sacrificed 72-96 hoursafter booster immunization. The blood was then collected and the spleenwas resected to prepare the suspension of the splenocyte (suspending inRPMI 1640 medium). The splenocytes were counted with a cell counter.Then the splenocytes were mixed in a 6:1 ratio with the SP2/0 mousemyeloma cells and centrifuged. The cells were fused with PEG(PEG 1500),then mixed with the equal volume of feeder cells, and transferred to96-well plate (200 uL/well). At the atmosphere of 5% CO₂, the 96-wellplate was incubated in a incubator (ESPEC BNA-31) at 37° C. 3 dayslater, half of the culture medium was replaced by flesh HT medium (1361mg hypoxanthn and 0.388 mg thymidine, with the addition of RPMI 1640medium (GIBCO Int.) to 100 mL, dissolved at about 45-50° C. andfiltrated for sterilization.). 7 days later, the 96-well plate wascoated with NE2, and ELISA assay was performed on the hybridoma cellculture as described below The cells positive to ELISA assay were clonedby limiting dilution means.

[0175] ELISA Assay

[0176] 100 uL NE2 were purified by HPLC described in Example 5 at 37°C., and then dissolved in 0.05 mol/L CB(20.02 g Na₂CO₃ and 2.52 gNaHCO₃, with the addition of ddH₂O to 1L, pH9.5) to a finalconcentration of 0.3 ug/mL. The 96-well polyvinyl microtiter plate wastreated with the resulting solution for 2 hours at 37° C. and thenovernight at 4° C. The microtiter plate was washed with PBST(8.0 g NaCl,0.2 g KH₂PO4, 2.9 g Na₂HPO₄ 12H₂O, 0.2 g KCl and 0.5 mL Tween-20, withthe addition of ddH₂O to 1L, pH7.4) to remove the unabsorbed antigens.Then 200 uL blocking solution (2% glutin, 0.2% casein and 2% sucrose in1×PBS) were added per well and incubated for 2 hours. Then pour off thesolution, dry the well and store in vacuum at 4° C.

[0177] To assay, 100 uL cell culture were added to each well, and setone positive control (add 100 uL 1:100 diluted polyclonal anti-NE2serum) and one negative control (add 100 uL HT medium) for each plate.After incubating at 37° C. for 30 min, the plate was washed with PBSTfor 5 times and then dried. HRP-GAM Ig (DAKO company) was added andincubated for anoth r 30 min at 37° C. The plate was washed withPBS-Tween-20 again for 5 times and dried 50 uL substrate solution A(13.42 g Na₂HPO₄ 12H₂O, 4.2 g citric acid H₂O and 0.3 g H₂O₂, with theaddition of ddH₂O to 700 mL) and 50 uL substrate solution B( 0.2 g TMDand 20 mL dimethylformamide, with the addition of ddH₂O to 700 mL) wereadded to the plate and incubated for 10 min at 37° C. 50 uL stopsolution was used to terminate the reaction. The OD₄₅₀ value of eachwell was read by an ELISA reader. In general, the OD450 value at leasttwice higher than that of the negative control can be considered aspositive.

[0178] The Preparation of the Ascites and the Purification of theMonoclonal Antibodies

[0179] Each 10-week-old Balb/C mouse was inoculated intraperitoneallywith 0.5 mL incomplete Freud's adjuvant. 2-7 days later, the hybridomacells were collected and centrifuged. Then discard the supernatant andadd serum-free medium to the cells to a final concentration of2×10⁵-2×10⁶ cells/mL. 0.5 mL resulting suspension of the cells was usedto inoculate each mouse. The ascites were harvested 7-10 days later whenthe abdomen of the mouse swelled, and then centrifuged for 15 min at3,000 rpm. The clear liquid in the middle part of the tube was pipettedout and filtered with 0.45 um Millipore membrane for sterilization. Thefiltrate was stored at −20° C.

[0180] Dilute the treated ascites with the equal volume of PBS (81 mL0.2 mol/L Na₂HPO₄ and 19 mL 0.2 mol/L NaH₂PO₄, with the addition ofnormal saline to 100 mL). (NH₄)₂SO₄ was then added dropwise with gentlystirring until 50% saturation, and kept at 4° C. overnight. The solutionwas centrifuged(12,000 rpm) at 4° C. for 15 min, and the supernatant wasdiscarded. The pellet was dissolved in PBS (2 volumes of the ascitesused). (NH₄)₂SO₄ was added dropwise again to the resulting solution withstirring until 33% saturation, and kept overnight at 4° C. The solutionwas centrifuged(12,000 rpm) at 4° C. for 15 min, and the supernatant wasdiscarded. The pellet was dissolved in PBS (2 volumes of the ascitesused). (NH₄)₂SO₄ was added dropwise with gently stirring until 50%saturation, and kept at 4° C. overnight. The solution wascentrifuged(12,000 rpm) at 4° C. for 15 min, and the supernatant wasdiscarded. The pellet was then dissolved in proper amounts of PBS in adialysis bag and dialyzed in 50-100 volumes of 120 mmol/L Tris-HClbuffer( containing 20 mmol/L NaCl, pH7.8) for about 12 hours at 4° C.with stirring. Replace the buffer for more than three times. Thedialysate was stored at −20° C.

[0181] According to the method described above, monoclonal antibodieswere prepared by immunizing Balb/C mice with polypeptide NE2 of theinvention, and 8 anti-NE2 monoclonal antibodies were identified(1F6,2C9, 3F5, 8C11, 8H3, 13D8, 15B2 and 16D7). Coat the eppendorf tubes withsaid 8 antibodies respectively, and test the capability of saidantibodies binding native HEV by capture RT-PCR(see Example 9). As aresult, 8C11, 8H3 and 13D8 shown significant activity of binding HEV,which indicated that their recognition sites were the native epitopes onthe surface of the viral coating. Said three antibodies were used inExample 10.

Example 9

[0182] Testing the Capability of mAb Binding HEV by Antibody-CapturingRT-PCR

[0183] The 1.5 mL eppendorfs were irradiated by ultraviolet for 30 min.and then added 500 uL mAb 1:1000 diluted in CB (20.02 g Na₂CO₃ and 2.52g NaHCO₃, with the addition of ddH₂O to 1L, pH9.5). After incubatingovernight at 37° C., pour off the buffer, and add 1.5 mL blockingbuffer(1×PBS with 2% albumin, pH7.4) to block 2 hours at 37° C. Thenpour off the blocking buffer and add 500 uL 10% dejecta in sterilizednormal saline which is positive for HEV. After reaction at 37° C. for 2hours, wash the eppendorf with PBST for 6 times and then add 250 uLddH₂O to each eppendorf. The RT-PCR assay was then performed accordingto Example 14. As a result, the monoclonal antibodies of 8C11, 8H3 and13D8 were capable of binding HEV, while 1F6, 2C9, 3F5, 15B2 and 16D7were not capable of binding HEV.

Example 10

[0184] ELISA of the Polypeptides of the Present Invention with the Serumfrom Positive Rhesus Monkey, Serum from Human and Murine DerivedMonoclonal Antibodies and the Dot Blotting of the Polypeptides of thePresent Invention with Murine Derived Monoclonal Antibodies

[0185] ELISA of the Recombinant Polypeptide with Serum from PositiveRheseus Monkey, Serum from Human and Marine Derived MonoclonalAntibodies

[0186] The polypeptides of the present invention shown in Table 2 areproduced and purified according to the methods mentioned in examples1-6. The resulted purified recombinant protein samples withconcentration of 1 mg/ml are diluted 1:500 with PBS buffer (20 Mm,pH7.4), and coated 100 μl/well on the 96-well microtitre plate under thefollowing condition: incubation at 37° C. for 2 hours and thenincubation overnight for about 12 hours at 4° C. After washing once withPBS-Tween20 washing solution (8.0 g NaCl, 0.2 g KH₂PO₄, 2.9 gNa₂HPO₄.12H₂O, 0.2 gKCl and 0.5 ml Tween20, adding non-ionic H₂O tofinal volume 1L, pH7.4) on the automatic washer (TECAN, M12/4R Columbusplus), and drying, blocking solution (2% glutin, 0.2% casein and 2%sucrose in PBS) was added, 200 μl/well, incubation at 37° C. for 30mins. Then the properly diluted anti-serum or monoclonal antibody wasadded, at 37° C. for 30 mins. After washing 5 times with PBS-Tween20washing solution on the automatic washer at 20 seconds interval anddrying, the properly diluted HRP-labelled second antibody (goatanti-human, mouse IgG antibody) was added, at 37° C. for 30 mins. Afterwashing 5 times with PBS-Tween20 washing solution on the automaticwasher at 20 seconds interval and drying, a drop of each of thechromogenic agent A and B (A: 13.42 g Na₂HPO₄.12H₂O, 4.2 g citric acidH₂O and 0.3 g H₂O₂, adjusting the volume to 700 ml with non-ionic water;B: 0.2 g TMB, 20 ml dimethylformamide, adjusting the volume to 700 mlwith non-ionic water) was added to develop color at 37° C. for 10 mins.A drop of the stop solution (2M H₂SO₄) was added The OD_(450nm) wasmeasured on a microplate reader (RECAN, Sunrise Remote/Touch Screen)(with reference wavelength of 620 nm). 3 times of the mean value of thenegative control was set as the positive threshold value, and the resultis positive when the OD value thereof is higher than the thresholdvalue.

[0187] Dot Blotting of the Polypeptides of the Present Invention withVarious Murine Derived Monoclonal Antibodies

[0188] 10 uL (1 mg/ml) of each of the polypeptides listed in Table 2which were produced according to the methods of examples 1-5 andpurified by HPLC gel filtration was dotted respectively and slowly onthe nitrocellulose membrane and air-dried. After blocking with 5% skimmilk for 1.5 hours at room temperature, various murine derivedmonoclonal antibodies produced as mentioned in example 8 (the cellsupernatant secreted by monoclonal B lymphocytes at 1:100 dilution with5% skim milk) were added, reacting at room temperature for 1 hour. Thenthe membrane was washed 3 times using TNT (10 mM Tris.Cl, pH8.0, 150 MmNaCl, 0.05% Tween20) at 5 mins interval. The HRP-labelled Goatanti-mouse IgG (produced by JINGMEI Biological Company, dilated in1:1000 with 5% skim milk) was added, and reacted at room temperature for1 hour. After washing 3 times with TNT at 5 mins interval, NBT/BCIP(C₄₀H₃₀N₁₀O₆Cl₂/C₈H₆BrClNO₄P.C₇H₉N) was added to develop color. The dotswere scanned with gel imagining system and diverted into the values ofgrey degree and divided into five positive grades as ++++, +++, ++, +,+− and negative grade as −. Compared with classic Western blotting, thismethod can reflect more really the immunoreactivity in the absence ofdenaturing agent due to not subject to be denatured with SDS. TABLE 4The reactivity of polypeptide of present invention against murinederived monoclonal antibody, serum from HEV patient in recovery phase,and serum from HEV-infected rhesus monkey in acute phase ELISA poly-monkey human dot blotting peptide serum serum 8C11 8H3 13D8 8C11 8H313D8 NE2 ++ ++ +++ ++ +++ ++++ ++ +++ 193C ++ ++ +++ + ++ ++ ++ ++178C + ++ +++ +− +++ ++ ++ ++ 168C − +− ++ − +++ +/− +/− +/− 158C +− + +− +++ + +/− +/− 148C + ++ − − − +/− +/− +/− 138C − − − − − +/− +/− +/−NE2I +++ ++ + +++ +++ +++ ++ +++ 217I + + ++ − ++ + + + 193I ++ ++ ++ −++ +++ ++ +++ 178I +++ ++ ++ − ++ ++ ++ ++ NE2D ++ ++ ++ − ++ + + + 217D++ ++ ++ − ++ + + + 193D ++ ++ ++ − ++ + + + 178D ++ ++ +++ − +++ + + +235N ++ +− +++ +− +++ +++ + +++ 225N +++ +++ +++ +− +++ ++++ + +++ 209N++ ++ +++ + +++ ++++ + +++ 208N + +++ +++ − +++ ++ + ++ 207N − − − − −++ + + 203N − − − − − ++ + + 193N − − − − − ++ + + 176N − + − − − ++/− + 247 +++ + +++ ++++ +++ ++++ 232 +++ + +++ ++++ ++++ ++++ 222 ++++− +++ ++++ ++ ++++ 205 ++ + +++ − +++ +++ ++ ++ 201 +++ + ++ ++++ ++++++ 189 − +− +++ − +++ ++ ++ ++ 188 − +− ++ − +++ ++ ++ ++ 183 − + +− −+− ++ ++ ++ 173 − − − − − ++ + ++ 170 +++ − +++ ++++ +++ ++++ C160 − − −− − − N160 ++ − ++ ++ − ++ 150 − − − − − − 144 ++ − ++ ++ − ++ 142 − − −− − − 134 − − − − − −

[0189] Result

[0190] Respectively, the purified recombinant polypeptides listed inTable 4 were coated on microtitre plate, and the reactivities thereof tothree murine derived monoclonal antibodies 8C11, 8H3 and 13D8 mentionedin example 8, serum of HEV patient in recovery phase and serum fromHEV-infected rhesus monkey in acute phase were examined by ELISA, andthe reactivities thereof to the used three monoclonal antibodies wereexamined by Dot blotting assay. The results showed that the polypeptidesNE2, 193C, 178C, NE2I, 235N, 225N, 209N, 247, 232, 222 and 201 hadbetter reactivities to each of the serum/monoclonal antibody. Thissuggested that the polypeptides had better native HEV epitope and can beused to diagnostic kit and/or vaccine for HEV.

[0191] The polypeptides 138C, C160, 150, 142 and 134 had poorreactivities to various antibodies. It showed that the formation of themajor native epitope ORF2 involved at least the fragment of aa469 toaa600.

[0192] The sequences of the polypeptides 170, C160, N160, 150, 144, 142and 134 were those that aa459-aa628, aa469-aa628, aa459-aa618,aa469-aa618, aa459-aa602, aa459-aa600 of the ORF2 were linkedrespectively to the initial amino acid (Met).

Example 11

[0193] The Western Blotting of the Polypeptides of the Present Inventionwith Murine Derived Monoclonal Antibodies

[0194] The Western blotting of the polypeptides 208N, 209N and 225N ofthe present invention produced as mentioned in examples 1-5 with murinederived monoclonal antibodies 1F6, 2C9, 3F5 produced in example 8 wascarried out. The said polypeptides were separated by SDS-PAGE, thentransferred to nitrocellulose membrane according to the conventionalmethods and 5% skim milk was added for blocking 1.5 hours; variousmurine derived monoclonal antibodies (the cell supernatant secreted bymonoclonal B lymphocytes at 1:100 dilution with 5% skim milk) wereadded, reacting at room temperature for 1 hour; after washing 3 times at5 mins interval, anti-mouse IgG labeled with HRP (at 1:1000 dilutionwith 5% skim mink) was added, room temperature for 1 hour, after washing3 times with TNT at 5 mins interval, NBT/BCIP were added to developedcolor. The results of Western blotting are shown in FIG. 6. The resultsshow that the polymer bands, in particular in lane 8, which cannot beobserved by the staining of Coomassie Brilliant Blue R250, can beobserved due to the enzyme-linked amplifying effect of Western blottingassay. It is further confirmed that the recombinant polypeptide 208N ofthe present invention does not form into polymer during 12% SDS-PAGE,and that the monoclonal antibody 1F6 has stronger activity to 209N and225N compared with the monoclonal antibodies 2C9 and 3F5.

Example 12

[0195] Preparation of the Vaccine Containing Polypeptide 201 and theAssay of the Immunizing Mouse with it

[0196] Preparation of the Vaccine Containing Polypeptide 201 withFreund's Adjuvant

[0197] The polypeptide201 of the present invention produced asabove-mentioned and purified by HPLC purity>95% and the concentration ofthe protein 1.02 mg/ml) was diluted with PBS, and the equivalent volumeof complete Freund's adjuvant (containing BCG) was added to reach adesired final concentration of the polypeptide201 (for example, if eachmouse was desired to be immunized 100 μl, 5 μg, the concentration of thepolypeptide 201 would be prepared into 0.05 mg/ml). The solution wasmixed and emulsified for 30 mins until no separating liquid phaseappeared after keeping still for 30 mins.

[0198] Using Freund's adjuvant as vaccine adjuvant, four groups mice(each group of 3 Kunming White mice), each was injected intramuscularlywith 0.5, 1, 2, 5 μg in 100 μl/mouse according to the immunizingschedule of 0, 7, 28 days. The results were shown in FIG. 7. The resultsindicated that ORF2-201 vaccine prepared with Freund's adjuvant withdoes above 2 μg had very strong immunogenicity, and the antibody wasstarted to produce at the second week after immunization and to reachthe highest titre at the forth week. It is thought that the antibodywith higher titre can be produced only in the mice immunized withprotein antigen at the does of 30-70 μg/mouse according to the commonbooks and literatures on immunology. Therefore, the results show thatthe vaccine, the polypeptide 201 of the present invention combined withFreund's adjuvant, has remarkably high immunogenic effect compared withthe available vaccine.

[0199] Preparation of the Vaccine Containing the Polypeptide 201 withAluminum Adjuvant

[0200] A desired amount of the original aluminum adjuvant (A13+13.68%,Na+3.36%, pH.5.55), which was from the Lanzhou Biological ProductInstitute of China, was adjusted with 1N NaOH till to produceprecipitate. Aft r mixing completely, 1×PBS was added to reach thedouble volume. Then centrifuge was performed at 10,000 for 1 min, andthe supernatant was discarded. The precipitate was resuspended with1×PBS to the double volume again, and centrifuged at 10,000 for 1 minand the supernatant was discarded. Such process was repeated severaltimes until the pH reached 7-7.4. Finally, the precipitate wasresuspended with equal volume of 1×PBS, and the solution was sterilizedand stored at 4° C., and used as 9×store solution.

[0201] Similarly, the polypeptide201 produced as above-mentioned andpurified by HPLC (purity>95% and the concentration of the protein 1.02mg/ml) was diluted with PBS, and the {fraction (1/9)} volume of aluminumadjuvant was added to reach a desired final concentration of thepolypeptide201, and mixed overnight at 4° C. The immunized does is 100μl/mouse. Using aluminum adjuvant as vaccine adjuvant, each group of 3Bal b/c mice, each was injected intramuscularly with 2, 5, 10 μg/mouseaccording to the immunizing schedule of 0, 7, 28 days. The results wereshown in FIG. 9. The results indicated that the does of 5, 10 μg/mousehas better immunogenic effect, which is comparable to that of theavailable HBV surface antigen vaccine currently, which is a particulateantigen and is thought to have better immunogenicity compared with themonomer antigen. Therefore, the results show that the polymer of thepresent invention is useful for the vaccine.

[0202] The Immunity Assay of the Polypeptide 201 without Adjuvant

[0203] The polypeptide 201 obtained as above-mentioned was solved in 4Murea, and the supernatant was dialyzed with PBS (pH7.45) to renature,with the purity of about 95%. Using Freund's adjuvant as vaccineadjuvant, each group of 3 Kunming White mice, each was injectedintramuscularly with 5, 25,50 μg/mouse (the control group with 5μg/mouse) according to the immunizing schedule of 0, 7, 28 days. Theresults were shown in FIG. 8. The results indicated that the remarkableantibodies were produced in the mice immunized with non-adjuvantORF2-201 vaccine, and the used dosage thereof was comparable to thatconventional antigen with Freund's adjuvant. It further shows that thepolymer polypeptide of the present invention has higher immunogenicitycompared with the conventional antigen.

[0204] It may be concluded from the above results that the Balb/c micecan be elicited to produce specific antibody when immunized with boththe recombinant polypeptide 201 and NE2 (5 μg/mouse), regardless of theformulation of Freund's adjuvant or aluminum adjuvant. And otherrenaturable polypeptides formulated with Freund's adjuvant can alsoelicit the mice to produce specific antibodies Therefore, thepolypeptides of the present invention have the good properties to use asvaccine.

Example 13

[0205] Immunization of Rhesus Monkeys with the Vaccine Comprising aRecombinant Polypeptide of the Present Invention

[0206] Six rhesus monkeys with normal ALT and negative HEV were selectedand divided into two groups, one group designated HF1, HF2 and HF3 andthe other group designated HF4, HF5 and HF6. The two groups of thesubject rhesus monkeys were vaccinated by intradeltoidal injection withthe aluminum adjuvant containing polypeptide NE2I vaccine andpolypeptide 201 vaccine prepared as described in the examples 1-5 and 12at the dosages of 20 μg, respectively. Such vaccinations were carriedout on days 0, 10, and 30 respectively. Three weeks after the lastvaccination, the titers of the anti-NE2I 1 gG antibody from the animalswere tested by indirect ELISA with the results as follows: HF1(1:16000),HF2(1:4000), HF3(1:8000), HF4(1:2000), HF5(1:3000) and HF6(1:5000).

[0207] The above results illustrated by polypeptides NE2I and 201indicated that the recombinant polypeptides of the invention have betterimmunogenicity compared with HEV ORF2 polypeptide trpE-C2 (amino acids225-660 of SEQ ID NO: 1) used as immunogen in U.S. Pat. No. 5885768. InU.S. Pat. No. 5885768, Reyes, et al. vaccinated 4 cynomolgus monkeys byintravenous injection with 50 ug of HEV ORF2 polypeptide trpE-C2expressed from E. coli in combination with an improved aluminum adjuvanton days 0 and 30 respectively. Two cynomolgus monkeys injected withadjuvant alone were used as control. No antibody to HEV was detected inthe group of the vaccinated monkeys 4 weeks after the secondvaccination. Two of these vaccinated monkeys were selected to receive athird vaccination with 80 ug of insoluble trpE-C2 polypeptide withoutadjuvant on day 58, and the anti-HBV antibody was detected only 4 weekslater.

Example 14

[0208] Challenging with HEV of the Rhesus Monkeys Immunized with VaccineComprising the Recombinant Polypeptide of the Present Invention

[0209] Preparation and Quantification of Hepatitis E Virus (HEV)

[0210] A fecal from a HEV patient from Xinjiang, China, was formulatedto 10% suspension in sterile physiological saline solution. Thesuspension was centrifuged at 12000 g for 20 minutes at 4° C., and thesupernatant was filter-sterilized with 0.2 μm sterile filter (NALGENE®Cat.No.190-2520). HEV in a PCR-detectable amount was used as aninfection dosage.

[0211] Extraction, reverse transcription and PCR of RNA of HEV fromfecal: HEV RNAs were extracted from the 10% fecal suspension usingTrizol reagent (GIBCOL) according to its manipulation instructions, andwere subjected to reverse transcription in a 20 μl reaction volume at42° C. for 40 minutes with the specific primer A3 (4566-4586,5′-ggctcaccggagtgtttcttc-3′) as RT primer using AMV reversetranscriptase. Then, the first round of RT-PCR was carried out in afinal volume of 20 ul using 2 ul RT product as template and using A3primer and A5 primer (4341-4362, 5′-ctttgatgacaccgtcttctcg-3′) under thefollowing reaction conditions: pre-denaturing at 94° C. for 5 min; 35cycles of denaturing at 94° C. for 40 s and extending at 68° C. for 40s; extending at 75° C. for 5 min. The second round of PCR was carriedout in a final volume of 20 ul using 2 ul of the first round reactionproduct as template and using primers B5 (4372-7392,5′-gccgcagcaaaggcatccatg-3′) and B3 (4554-4575,5′-gtgtttcttccaaaaccctcgc-3′) under the following reaction conditions:pre-denaturing at 94° C. for 5 min; 35 cycles of denaturing at 94° C.for 40 s, annealing at 56° C. for 40 s and extending at 72° C. for 1 min20 sec; extending at 75° C. for 5 min.

[0212] Grouping of rhesus monkeys used in this experiment: immunizedgroup 1 including rhesus monkeys V10, V11 and V12 corresponding toanimals HF1, HF2 and HF3 in Example 2, respectively; immunized group 2including rhesus monkeys V13, V14 and V15 corresponding to animals HF4,HF5 and HF6 in Example 2, respectively; a control group including threenon-irnmunized rhesus monkeys designated V16, V17 and V18.

[0213] Challenging with HEV

[0214] HEV in a PCR-detectable amount was used as one infection dosage.Three weeks after the last vaccination of the rhesus monkey HF1-6 withvaccine comprising the recombinant polypeptides of the present inventionas described in Example 13, the above monkeys were challenged with 1,000infection dosages of HEV. After challenge ALT of every monkey did notincrease. Anti-NE2I-IgG in animals V16 and V17 was detected at week 4,and Anti-NE2-IgG was detected in animal V18 at week 5. From days 1 to37, no HEV excretion was detected in animals V10-15; HEV excretion inanimal V16 started on day 5 and ended on day 30; and HEV excretion inanimals V17 and V18 began on day 5 too, but didn't stop on day 37. Theseresults indicated that the polypeptide of the present invention asvaccine possessed better immunogenicity and produced better protectioncompared with the polypeptide trpE-C2 of HEV ORF2 in U.S. Pat. No.5885768.

[0215] From these results, it could be seen that when low dosage ofvaccine of this invention was used to vaccinate monkeys, the vaccinatedmonkeys could produce excellent antibody response to HEV and abnormalALT and virus excretion in fecal were not observed after challenge withHEV. Thus better immunoprotection was produced compared with thereported vaccination results of the vaccines prepared using polypeptidetrpE-C2 from HEV ORF2 (U.S. Pat. No. 5885768) and of the vaccinescomprising the polypeptides prepared by Tsarev et al. In addition, thebaculovirus expression system applied by Tsarev and Genelabs co. haspotential harn to the human body, so there was no report hitherto thatany recombinant protein expressed by this system was approved as acommercialized drug or a commercialized vaccine used in human. On thecontrary, several of the recombination proteins expressed by E.coliexpression system according to the present invention were approved ascommercialized in vivo drugs used in human, and have more reliablesafety.

Example 15

[0216] Preparation of a Chimeric Polypeptide Comprising Polypeptide 247of the Invention Linked with the Epitope in HEV ORF3

[0217] PCR reaction was performed with the full length genrome ofhepatitis E virus (HEV) isolated from a HEV patient of XinJiang, China.(Aye,T. T.,Uchida et al. Nucleic Acids Research, 20(13), 3512(1992);GenBank accession number: GI221701) as template using the forwardprimer, 372FP (5′-ggatcccatatgaataacatgtcttttgct-3′), introducingrestricted endonuclease sites BamHI and NdeI at its 5′-terminal, and thereverse primer, 372BRP (5′-ggatcotcggcgcggcc-3′), introducing arestricted endonuclease site BamHI in its 5′-terminal, under thefollowing reaction condition: 94° C. 5 min, 30 cycles of 94° C. 50 sec,56° C. 50 sec and 72° C. 30 sec, and 70° C. 1 min. A specific DNAfragment with the size of about 370 bp encoding the epitode in HEV ORF3was obtained. The PCR product obtained above was ligated into commercialpMD 18-T plasmid (TAKARA co.). A positive sub-clone in which the epitopegene int HEV ORF3 was inserted was identified by digestion with BamHI.DNA sequencing indicated no mutation in the clone, and thereby the aminoacid sequence of the epitope gene in HEV ORF3 was obtained, as set forthin SEQ ID NO: 11.

[0218] The HEV-ORF3 gene fragment was obtained by digestion with BamHI,and ligated into pTO-T7-ORF2-247 expression plasmid vector (preparedaccording to the method described in Example 1) which had been digestedwith BainHI. A positive expression clone, pTO-T7-ORF3-247, in which theHEV ORP3 gene fragment was inserted, was identified by digestion withBamHI The clone was transformed into E.coli ERR2566 strain, which wasused to express the ORF3-247 chimeric polypeptide.

Example 16

[0219] Preparation of a Chimeric Polypeptide of NE2D Linked withHemagglutin Antigen from Influenza Virus

[0220] The nucleotide sequence of the chimeric peptide was obtained byPCR amplification with a HEV-ORF2 mutant sequence (SEQ ID NO: 6)prepared in example 1 as template by using primer pairs HAFP/E2RD. ThePCR reaction is carried out as follows: pre-heating at 94° C. for 5minutes; 30 cycles of denaturation at 94° C. for 50 seconds, annealingat 56° C for 50 seconds, extension at 72° C. for 70 seconds; andextension at 72° C. for 10 minutes at last. The resulted PCR product isan about 800 bp DNA fragment, encoding chieric polypeptide comprising HAfrom influenza virus and polypeptide NE2D of present invention. Theforward primer HAFP contains BamHI and NdeI restriction sites. Thereverse primer E2RD contained EcoRI restriction site and a stoptranslation codon TAA. The sequence for NdeI site is CAT ATG, whereinATG is an initial translation codon. Moreover, in order to maintain theexact conformations of the two peptides HA and NE2D, respectively, aflexible linker Gly-Gly-Ser coded by CAG CTG TTC was introduced betweenHA and NE2D peptides. Therefore, the chimeric polypeptide HA-NE2D couldbe suitably employed in HEV vaccine. The sequences of primer pairs areas follows; HAFP:5′-AGA TCT  CAT ATG  TCT  AAA  GCT TTC TCT  AAC TGC TAC CCT   BglII     NdeI 91 Ser  Lys  Ala Phe  Ser  Asn Cys  Tyr Pro   TAC GAC GTT CCG GAC TAC GCT TCT TTA     GGT GGA TCC   Tyr Asp Val Pro Asp Tyr Ala Ser  Leu108 Gly Gly Ser   CAG CTG TTC TAC TCT CGT CC-3′ E2RD: 5′-gaattcttagggggctaaaacagc-3′

[0221] Preparation of expression vector comprising a nucleic acidconstruction encoding resulted chimeric polypeptide

[0222] The aforementioned PCR product was cloned into commercialavailable plasmid pMD 18-T (TAKARA com. Ltd.). The interest sequencethen was acquired by BamHI/EcoRI digestion from pMD 18-T-HA-ORF2- NE2Dplasmid, and integrated into expression vector with BamHI/EcoRIdigestion and ligation. An HA-ORF2-NE2D chimeric peptide was isolatedfrom E.coli ERR2566 host cells transformed with the pTO-T7-HA-ORF2-NE2Dexpression plasmid. The amino acid sequence was designated as SEQ ID NO:12.

Example: 17

[0223] Detecting IgG for HEV in Biological Samples with the IndirectElisa Kit Based on Polypeptide NE2I of this Invention

[0224] The kit detecting IgG for HEV with polypeptide NE2I of thisinvention comprises: microtiter plate coated with recombinationpolypeptide NE2I and blocked with blocking solution (20 mM pH7.2 PB,0.5% Casein, 2% Gelatin); sample diluent (20 mM pH7.2 PB, 1% Casein);working conjugate (goat anti-human IgG (DAKO) labeled with HRP dilutedwith enzyme diluent (20 mM pH7.2 PB, 0.5% Casein, 10% NBS));nonbioactivity material such as 20×PBST, chromatogen A, chromatogen Band stop solution (Beijing wantai).

[0225] Series of monkeys' sera were detected with anti-REV IgG kit basedon NE2I compared with two commercial anti-HEV IgG kits from Beijingwantai and Singapore Genelabs. The monkeys' sera were the sera ofmonkeys No1, No2, No3 and No13 mocking natural infected monkey by HEVfrom 0 to 18 weeks after intravenous challenge.

[0226] The manipulating procedure is as follows: add 100 ul of samplesoluent to each microtiter; add 10 ul of specimen to the microtiter; mixthoroughly by tapping gently on all sides of rriicroplate; Incubate for30 minutes at 37° C.; wash the microplate with 1 times. PBST five times;blot dry by inverting the microplate and tapping firmly onto absorbentpaper; add 100 ul of working conjugate to all wells and incubating themicroplate for 30 minutes at 37° C.; wash the microplate five timesagain and blotting dry; add 50 ul of chromatogen A and 50 ul ofchromatogen B and mixing thoroughly by tapping gently; incubate themicroplate for 10 minutes at 37° C. in dark; add 50 ul of stop solutionto each well and mixing gently by tapping the plate; determine theabsorbance for each well at 450 nm/620 nm. The two commercial anti-HPVIgG kits was assayed accurately according to each assay procedure.

[0227] The result is as follows: the kit based on NE2I detectedseroconversion earlier than the two commercial kits from Beijing wantaiand Singapore Genelabs by 7-14 days; the duration of anti-NE2I-TgG waslonger than Genelabs anti-HEV-IgG and Wantai anti-HEV-IgG; thedetectable rate of anti-NE21-IgG was higher than the two commercialanti-HEV-IgG kits (see FIG. 10, raw data shown in table 5). When 34random sera of normal people were detected, the positive rates ofanti-NE21-IgG and of Genelabs anti-HEV-IgG were 35% and 11%respectively, and the later was absolutely included in the former. When263 clinical sera of hepatitis patients, the positive rates ofanti-NE2I-IgG and of Wantai anti-HEV-IgG were 27.2% and 10.6%respectively. When 91 sera of non-A, non-B, non-C hepatitis patients,the positive rates of anti-NE2I-IgG and of Genelabs anti-HEV-IgG were69.2% and 24.2% respectively. In a word, the anti-HEV-IgG based on NE2Iof this invention is better sensitive than the commercial anti-HEV-IgGkits. TABLE 5 Comparison of sensitivity for detection HEV infectedmonkey serum by anti-HEV IgG antibody detection kit of present invention(NE2I-IgG) and by two commercial available detection kit (Genelabs;WANTAI) Serum No. 1 0w 1w 2w 3w 4w 5w 6w 7w 8w 9w Genelabs Anti-HEV-IgG0.01 0.01 0.01 0.01 0.21 2.61 3 3 3 2.61 WANTAI Anti-HEV-IgG 0.01 0.020.01 0.02 0.01 1.82 2.45 2.48 2.42 1.7 inventive NE2I-IgG 0.01 0.02 0.011.87 2.56 2.5 2.52 2.49 2.72 2.52 Serum No. 1 10w 11w 12w 13w 14w 15w16w 17w 18w Genelabs Anti-HEV-IgG 1.78 1.36 0.92 0.92 0.92 0.96 0.72WANTAI Anti-HEV-IgG 1.37 0.54 0.36 0.25 0.18 0.1 0.05 0.05 0.03inventive NE2I-IgG 2.52 2.47 2.53 2.58 2.64 2.65 2.40 2.36 2.35 SerumNo. 2 0w 1w 2w 3w 4w 5w 6w 7w 8w 9w Genelabs Anti-HEV-IgG 0.05 0.07 0.050.04 3 3 3 3 3 3 WANTAI Anti-HEV-IgG 0.02 0.02 0.02 0.02 2.05 2.58 2.422.46 2.65 2.5 inventive NE2I-IgG 0.06 0.06 0.06 0.77 2.26 2.36 2.5 2.562.62 2.65 Serum No. 2 10w 11w 12w 13w 14w 15w 16w 17w 18w GenelabsAnti-HEV-IgG 3 3 3 3 3 3 3 WANTAI Anti-HEV-IgG 2.33 2.23 2.3 2.21 2.22.26 2.26 2.38 2.46 inventive NE2I-IgG 2.49 2.47 2.4 2.54 2.51 2.38 2.292.34 2.33 Serum No. 3 0w 1w 2w 3w 4w 5w 6w 7w 8w 9w GenelabsAnti-HEV-IgG 0.05 0.02 0.02 0.02 0.78 3 3 2.91 2.83 2.77 WANTAIAnti-HEV-IgG 0 0 0 0.02 0.02 1.87 1.62 1.67 1.82 1.48 inventive NE2I-IgG0.01 0.05 0.04 0.83 2.2 2.63 2.59 2.62 2.75 3 Serum No. 3 10w 11w 12w13w 14w 15w 16w 17w 18w Genelabs Anti-HEV-IgG 2.22 1.95 1.79 1.54 1.421.42 1.38 WANTAI Anti-HEV-IgG 1.05 0.67 0.75 0.76 0.58 0.38 0.46 0.340.23 inventive NE2I-IgG 2.7 2.6 2.55 2.6 2.54 2.51 2.51 2.36 2.51 SerumNo. 13 0w 1w 2w 3w 4w 5w 6w 7w 8w 9w Genelabs Anti-HEV-IgG 0.04 0.030.03 0.03 0.04 0.05 0.15 0.1 0.08 0.06 WANTAI Anti-HEV-IgG 0.02 0 0.010.01 0.01 0 0.01 0.02 0.04 0.03 inventive NE2I-IgG 0.1 0.11 0.09 0.092.3 2.45 2.51 2.5 2.34 2.21 Serum No. 13 10w 11w 12w 13w 14w 15w 16w 17w18w Genelabs Anti-HEV-IgG 0.06 0.07 0.08 0.07 0.1 0.15 0.07 0.04 0.02WANTAI Anti-HEV-IgG 0.03 0.03 0.02 0.02 0.02 0.02 0.02 inventiveNE2I-IgG 2.13 2.14 2.05 2.01 1.82 1.87 1.79 1.7 1.6

Example 18

[0228] Method for Labeling the Recombinant Proteins of Present Inventionwith HRP

[0229] Dissolving 1 mg of HRP (Biozyme R/Z>3) and NaIO4, respectively,in ultra-pure water (UPW); adding drop-wisely NaIO4 solution withagitation; the mixture solution is stand for 30 minutes in dark at roomtemperature; stepwise add 100 ul of 1% ethylene glycol solution, mixing;allow it to stand for 30 minutes in dark at 4° C. Dialyze recombinationprotein against carbonate buffer (10 mM pH9.6) 3 hours; add appropriatedialyzed recombination protein to the oxygenized HRP, dialyze for 6hours at room temperature (or 4° C.) in carbonate buffer (10 mM pH9.5)with gently stirring; add 20 ul of freshly prepared 0.1M NaBH4 solutionto the above-mentioned blending, allow it to stand for 2 hours at 4° C.in darkness, gently vortex once each 30 minutes; dialyze it in PBS (10mM pH7.2) overnight at 4° C.

Example 19

[0230] Diagnostic Kit for Detecting Antibody IgM against HEV inBiological Samples and Capture Assay for Detecting Antibody IgM againstHEV in Biological Aamples

[0231] Polypeptide 225N was labeled with HRP according to the methoddescribed in example 18.

[0232] The diagnostic kit for detecting antibody IgM against HEVcontaining HRP-labeled polypeptide NE2I of present invention comprises:microtiter strip that is pre-coated with mouse anti-human IgM μ chainpolyclonal antibody (Dako company, Denmark) and blocked with blockingsolution; sample diluent (20 mM pH7.2 PB, 1% Casein); working conjugate(HRP-labeled polypeptide 225N that is suitably diluted with enzymediluent (20 mM pH7.2 PB, 0.5% Casein, 10% NBS)); non-bioactivitymaterial, such as 20×PBST, chroinatogen agent A, chromatogen agent B andstop solution (WANTAI cotnpany, Beijing).

[0233] The capture assay using present diagnostic kit is carried out asfollows: adding 100 ul of diluent buffer to each well which ispre-coated with mouse anti-human IgM μ chain polyclonal antibody; adding1 ul of sample to be detected into the diluent buffer; mixing thoroughlyby tapping gently; incubating for 30 minutes at 37° C.; washing withPBST for five times; blot dry by inverting the microplate up-side-downand tapping finnly onto tissue; adding 100 ul of working conjugate(HRP-labeled polypeptide 225N that is suitably diluted) to each well andincubating the microplate for 30 minutes at 37° C.; washing five timesagain and blotting dry; then adding 50 ul of chromatogen A and 50 ul ofchromatogen B and mixing thoroughly by tapping gently; incubating for 10minutes at 37° C. in dark; add 50 ul of stop solution to each well andmixing gently by tapping the plate; determine the absorbance for eachwell at OD_(450nm/620nm).

[0234] Using the diagnostic anti-HEV-IgM kit as prepared in accordancewith present invention, 263 clinical sera of hepatitis patients weredetected by aforementioned capture assay with the positive rate of 11%;and 91 sera of non-A, non-B, non-C hepatitis patients were also detectedby said capture assay with the positive rate of 48.4%. Meanwhile, thepositive rate for these 91 sera sample detected by diagnostic kit ofanti-HEV-IgG from Genelabs was merely 24.2%. As indicated by the aboveresults, the positive sample detected by diagnostic anti-HEV-IgM kit ofpresent invention using capture assay is in good accordance with theclinical diagnosis. Moreover, most of positive samples detected byGenelabs anti-HEV-IgG kit are also positive in the present captureassay. That is to say, the anti-HEV-IgM kit of present invention as wellas said capture assay possess higher sensitivity and specificity inclinical HEV diagnosis than that of existing commercial available kits.

Example 20

[0235] Diagnostic Kit for Detecting Total Antibodies against HEV inBiological Samples and the Method for Detecting Total Antibodies againstHEV in Biological Samples

[0236] Polypeptide 225N was labeled with horseradish peroxidase (HEP)according to the method described in example 18.

[0237] The kit detecting total antibodies for HEV containingpolypeptides NE21 and 225N of present invention comprises: microtiterstrip pre-coated with recombination polypeptide NE2I and blocked withblocking solution (20 mM pH7.2 PB, 0.5% Casein, 2% Gelatin); workingconjugate (polypeptide 225N labeled with HRP diluted with enzyme diluent(20 mM pH7.2 PB, 0.5% Casein, 10% NBS)); non-bioactivity material, suchas 20×PBST, chromnatogen agent A, chromatogen agent B and stop solution(WANTAI company, Beijing).

[0238] The detecting protocol of present kit is as follows: adding 50 ulof serum and 50 ul of suitable diluted recombinant polypeptide 225N thatis HRP labeled into the microtiter on said strip which is in advancecoated with polypeptide NE2I; mixing by tapping gently on all sides ofmicro-plate; incubating for 60 minutes at 37° C.; washing themicro-plate with PBST for five times; blot dry by inverting themicro-plate; adding 50 ul of chromatogen A and 50 ul of chromatogen B;and then incubating the microplate for 15 minutes at 37° C.; finally,adding 50 ul of stop solution to each well and mixing gently by tappingthe plate; determine the absorbance for each well at OD_(450nm/620nm).

[0239] The detection results is provided as follows: detected with thesandwich total anti-HEV antibodies kit based on polypeptides NE21 and225N of present invention, 263 clinical sera from hepatitis patientswere detected with the positive rate of 52%, while only 10.6% isdetermined by detecting with WANTAI aniti-HEV-IgG kit. And further 91sera of non-A, non-B, non-C hepatitis patients were detected using thesame sandwich kit, with the positive rate of 54.9%. However, in these 91sera, the positive rate detected by Genelabs anti-HEV-IgG was merely24.2%. As indicated by the above data, detection with the presentdiagnostic kit is superior to that of existing commercial available kitsin clinical diagnosis.

1 12 1 660 PRT hepatitis E virus 1 Met Arg Pro Arg Pro Ile Leu Leu LeuLeu Leu Met Phe Leu Pro Met 1 5 10 15 Leu Pro Ala Pro Pro Pro Gly GlnPro Ser Gly Arg Arg Arg Gly Arg 20 25 30 Arg Ser Gly Gly Ser Gly Gly GlyPhe Trp Gly Asp Arg Val Asp Ser 35 40 45 Gln Pro Phe Ala Ile Pro Tyr IleHis Pro Thr Asn Pro Phe Ala Pro 50 55 60 Asp Val Thr Ala Ala Ala Gly AlaGly Pro Arg Val Arg Gln Pro Ala 65 70 75 80 Arg Pro Leu Gly Ser Ala TrpArg Asp Gln Ala Gln Arg Pro Ala Ala 85 90 95 Ala Ser Arg Arg Arg Pro ThrThr Ala Gly Ala Ala Pro Leu Thr Ala 100 105 110 Val Ala Pro Ala His AspThr Pro Pro Val Pro Asp Val Asp Ser Arg 115 120 125 Gly Ala Ile Leu ArgArg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr 130 135 140 Ser Ser Val AlaThr Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu 145 150 155 160 Ser ProLeu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala 165 170 175 ThrGlu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Ala Arg Ala Thr Ile 180 185 190Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser 195 200205 Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met 210215 220 Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile225 230 235 240 Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr ArgAsn Gln 245 250 255 Gly Trp Arg Ser Val Glu Thr Ser Gly Val Ala Glu GluGlu Ala Thr 260 265 270 Ser Gly Leu Val Met Leu Cys Ile His Gly Ser ProVal Asn Ser Tyr 275 280 285 Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly LeuLeu Asp Phe Ala Leu 290 295 300 Glu Leu Glu Phe Arg Asn Leu Thr Pro GlyAsn Thr Asn Thr Arg Val 305 310 315 320 Ser Arg Tyr Ser Ser Thr Ala ArgHis Arg Leu Arg Arg Gly Ala Asp 325 330 335 Gly Thr Ala Glu Leu Thr ThrThr Ala Ala Thr Arg Phe Met Lys Asp 340 345 350 Leu Tyr Phe Thr Ser ThrAsn Gly Val Gly Glu Ile Gly Arg Gly Ile 355 360 365 Ala Leu Thr Leu PheAsn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro 370 375 380 Thr Glu Leu IleSer Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro 385 390 395 400 Val ValSer Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val 405 410 415 GluAsn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp 420 425 430Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu 435 440445 Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val 450455 460 Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr465 470 475 480 Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val Tyr ValSer Asp 485 490 495 Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln AlaVal Ala Arg 500 505 510 Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly ArgPro Leu Ser Thr 515 520 525 Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val LeuPro Leu Arg Gly Lys 530 535 540 Leu Ser Phe Trp Glu Ala Gly Thr Thr LysAla Gly Tyr Pro Tyr Asn 545 550 555 560 Tyr Asn Thr Thr Ala Ser Asp GlnLeu Leu Val Glu Asn Ala Ala Gly 565 570 575 His Arg Val Ala Ile Ser ThrTyr Thr Thr Ser Leu Gly Ala Gly Pro 580 585 590 Val Ser Ile Ser Ala ValAla Val Leu Ala Pro His Ser Val Leu Ala 595 600 605 Leu Leu Glu Asp ThrMet Asp Tyr Pro Ala Arg Ala His Thr Phe Asp 610 615 620 Asp Phe Cys ProGlu Cys Arg Pro Leu Gly Leu Gln Gly Cys Ala Phe 625 630 635 640 Gln SerThr Val Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys 645 650 655 ThrArg Glu Leu 660 2 214 PRT hepatitis E virus 2 Met Gln Leu Phe Tyr SerArg Pro Val Val Ser Ala Asn Gly Glu Pro 1 5 10 15 Thr Val Lys Leu TyrThr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly 20 25 30 Ile Ala Ile Pro HisAsp Ile Asp Leu Gly Glu Ser Arg Val Val Ile 35 40 45 Gln Asp Tyr Asp AsnGln His Glu Gln Asp Arg Pro Thr Pro Ser Pro 50 55 60 Ala Pro Ser Arg ProPhe Ser Val Leu Arg Ala Asn Asp Val Leu Trp 65 70 75 80 Leu Ser Leu ThrAla Ala Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser 85 90 95 Thr Gly Pro ValTyr Val Ser Asp Ser Val Thr Leu Val Asn Val Ala 100 105 110 Thr Gly AlaGln Ala Val Ala Arg Ser Leu Asp Trp Thr Lys Val Thr 115 120 125 Leu AspGly Arg Pro Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe 130 135 140 PheVal Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr 145 150 155160 Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln 165170 175 Leu Leu Val Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr180 185 190 Thr Thr Ser Leu Gly Ala Gly Pro Val Ser Ile Ser Ala Val AlaVal 195 200 205 Leu Ala Pro Pro Pro Arg 210 3 194 PRT hepatitis E virus3 Met Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro 1 5 1015 His Asp Ile Asp Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp 20 2530 Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg 35 4045 Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr 50 5560 Ala Ala Glu Tyr Asp Gln Ser Thr Tyr Gly Ser Ser Thr Gly Pro Val 65 7075 80 Tyr Val Ser Asp Ser Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln 8590 95 Ala Val Ala Arg Ser Leu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg100 105 110 Pro Leu Ser Thr Ile Gln Gln Tyr Ser Lys Thr Phe Phe Val LeuPro 115 120 125 Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr LysAla Gly 130 135 140 Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln LeuLeu Val Glu 145 150 155 160 Asn Ala Ala Gly His Arg Val Ala Ile Ser ThrTyr Thr Thr Ser Leu 165 170 175 Gly Ala Gly Pro Val Ser Ile Ser Ala ValAla Val Leu Ala Pro Pro 180 185 190 Pro Arg 4 389 PRT hepatitis E virus4 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser His Met 1 5 1015 Asn Asn Met Ser Phe Ala Ala Pro Met Gly Ser Gln Pro Cys Ala Leu 20 2530 Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg 35 4045 His Arg Pro Val Ser Arg Leu Ala Ala Val Val Gly Gly Ala Ala Ala 50 5560 Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Pro Ser Pro Ser 65 7075 80 Gln Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser Pro 8590 95 Leu Arg Pro Gly Leu Asp Leu Val Phe Ala Asn Pro Pro Asp His Ser100 105 110 Ala Pro Leu Gly Val Thr Arg Pro Ser Ala Pro Pro Leu Pro HisVal 115 120 125 Val Asp Leu Pro Gln Leu Gly Pro Arg Arg Gly Ser His MetThr Ser 130 135 140 Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile ProHis Asp Ile 145 150 155 160 Asp Leu Gly Glu Ser Arg Val Val Ile Gln AspTyr Asp Asn Gln His 165 170 175 Glu Gln Asp Arg Pro Thr Pro Ser Pro AlaPro Ser Arg Pro Phe Ser 180 185 190 Val Leu Arg Ala Asn Asp Val Leu TrpLeu Ser Leu Thr Ala Ala Glu 195 200 205 Tyr Asp Gln Ser Thr Tyr Gly SerSer Thr Gly Pro Val Tyr Val Ser 210 215 220 Asp Ser Val Thr Leu Val AsnVal Ala Thr Gly Ala Gln Ala Val Ala 225 230 235 240 Arg Ser Leu Asp TrpThr Lys Val Thr Leu Asp Gly Arg Pro Leu Ser 245 250 255 Thr Ile Gln GlnTyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly 260 265 270 Lys Leu SerPhe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr 275 280 285 Asn TyrAsn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn Ala Ala 290 295 300 GlyHis Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly 305 310 315320 Pro Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro His Ser Ala Leu 325330 335 Ala Leu Leu Glu Asp Thr Met Asp Tyr Pro Ala Arg Ala His Thr Phe340 345 350 Asp Asp Phe Cys Pro Glu Cys Arg Pro Leu Gly Leu Gln Gly CysAla 355 360 365 Phe Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Met LysVal Gly 370 375 380 Lys Thr Arg Glu Leu 385 5 1990 DNA hepatitis E virus5 atgcgccctc ggcctatttt gctgttgctc ctcatgtttc tgcctatgct gcccgcgcca 60ccgcccggtc agccgtctgg ccgccgtcgt gggcggcgca gcggcggttc cggcggtggt 120ttctggggtg accgggttga ttctcagccc ttcgcaatcc cctatattca tccaaccaac 180cccttcgccc ccgatgtcac cgctgcggcc ggggctggac ctcgtgttcg ccaacccgcc 240cgaccactcg gctccgcttg gcgtgaccag gcccagcgcc ccgccgttgc ctcacgtcgt 300agacctacca cagctggggc cgcgccgcta accgcggtcg ctccggccca tgacaccccg 360ccagtgcctg atgttgactc ccgcggcgcc atcctgcgcc ggcagtataa cctatcaaca 420tctcccctta cttcttccgt ggccaccggt acaaacttgg ttctatacgc cgctcctctt 480agcccacttc tacccctcca ggacggcacc aatactcata taatggccac agaagcttct 540aattatgccc agtaccgggt tgctcgtgcc acaattcgct accgcccgct ggtccccaac 600gctgttggtg gctacgccat ctccatctcg ttctggccac agaccaccac caccccgacg 660tccgttgaca tgaattcaat aacctcgacg gatgttcgta ttttagtcca gcccggcata 720gcctccgagc ttgttatccc aagtgagcgc ctacactacc gtaaccaagg ttggcgctct 780gttgagacct ccggggtggc ggaggaggag gccacctctg gtcttgttat gctctgcata 840catggctcac ctgtaaattc ttatactaat acaccttata ccggtgccct cgggctgttg 900gactttgccc tcgaacttga gttccgcaac ctcacccccg gtaataccaa cacgcgggtc 960tcccgttact ccagcactgc ccgtcaccgc cttcgtcgcg gtgcagatgg gactgccgag 1020cttaccacca cggctgctac ccgcttcatg aaggacctct attttactag tactaatggt 1080gtcggtgaga tcggccgtgg gatagcgctt accctgttta accttgctga caccctgctt 1140ggcggtctac cgacagaatt gatttcgtcg gctggtggcc agctgttcta ctctcgtccc 1200gtcgtctcag ccaatggcga gccgactgtt aagctttata catctgtaga gaatgctcag 1260caggataagg gtattgcaat cccgcatgac atcgacctcg gggagtctcg tgtagttatt 1320caggattatg acaaccaaca tgagcaggac cgaccgacac cttccccagc cccatcgcgc 1380cctttttctg tcctccgagc taatgatgtg ctttggcttt ctctcaccgc tgccgagtat 1440gaccagtcca cttacggctc ttcgaccggc ccagtctatg tctctgactc tgtgaccttg 1500gttaatgttg cgaccggcgc gcaggccgtt gcccggtcac tcgactggac caaggtcaca 1560cttgatggtc gccccctttc caccatccag cagtattcaa agaccttctt tgtcctgccg 1620ctccgcggta agctctcctt ttgggaggca ggtactacta aagccgggta cccttataat 1680tataacacca ctgctagtga ccaactgctc gttgagaatg ccgctgggca tcgggttgct 1740atttccactt acaccactag cctgggtgct ggtcccgtct ctatttccgc ggttgctgtt 1800ttagcccccc actccgcgct agcattgctt gaggatacca tggactaccc tgcccgcgcc 1860catactttcg atgacttctg cccggagtgc cgcccccttg gcctccaggg ctgtgctttt 1920cagtctactg tcgctgagct tcagcgcctt aagatgaagg tgggtaaaac tcgggagtta 1980tagtttattt 1990 6 1989 DNA hepatitis E virus 6 atgcgccctc ggcctattttgctgttgctc ctcatgtttc tgcctatgct gcccgcgcca 60 ccgcccggtc agccgtctggccgccgtcgt gggcggcgca gcggcggttc cggcggtggt 120 ttctggggtg accgggttgattctcagccc ttcgcaatcc cctatattca tccaaccaac 180 cccttcgccc ccgatgtcaccgctgcggcc ggggctggac ctcgtgttcg ccaacccgcc 240 cgaccactcg gctccgcttggcgtgaccag gcccagcgcc ccgccgttgc ctcacgtcgt 300 agacctacca cagctggggccgcgccgcta accgcggtcg ctccggccca tgacaccccg 360 ccagtgcctg atgttgactcccgcggcgcc atcctgcgcc ggcagtataa cctatcaaca 420 tctcccctta cttcttccgtggccaccggt acaaacttgg ttctatacgc cgctcctctt 480 agcccacttc tacccctccaggacggcacc aatactcata taatggccac agaagcttct 540 aattatgccc agtaccgggttgctcgtgcc acaattcgct accgcccgct ggtccccaac 600 gctgttggtg gctacgccatctccatctcg ttctggccac agaccaccac caccccgacg 660 tccgttgaca tgaattcaataacctcgacg gatgttcgta ttttagtcca gcccggcata 720 gcctccgagc ttgttatcccaagtgagcgc ctacactacc gtaaccaagg ttggcgctct 780 gttgagacct ccggggtggcggaggaggag gccacctctg gtcttgttat gctctgcata 840 catggctcac ctgtaaattcttatactaat acaccttata ccggtgccct cgggctgttg 900 gactttgccc tcgaacttgagttccgcaac ctcacccccg gtaataccaa cacgcgggtc 960 tcccgttact ccagcactgcccgtcaccgc cttcgtcgcg gtgcagatgg gactgccgag 1020 cttaccacca cggctgctacccgcttcatg aaggacctct attttactag tactaatggt 1080 gtcggtgaga tcggccgtgggatagcgctt accctgttta accttgctga caccctgctt 1140 ggcggtctac cgacagaattgatttcgtcg gctggtggcc agctgttcta ctctcgtccc 1200 gtcgtctcag ccaatggcgagccgactgtt aagctttata catctgtaga gaatgctcag 1260 caggataagg gtattgcaatcccgcatgac atcgacctcg gggagtctcg tgtagttatt 1320 caggattatg acaaccaacatgagcaggac cgaccgacac cttccccagc cccatcgcgc 1380 cctttttctg tcctccgagctaatgatgtg ctttggcttt ctctcaccgc tgccgagtat 1440 gaccagtcca cttacggctcttcgaccggc ccagtctatg tctctgactc tgtgaccttg 1500 gttaatgttg cgaccggcgcgcaggccgtt gcccggtcac tcgactggac caaggtcaca 1560 cttgatggtc gccccctttccaccatccag cagtattcaa agaccttctt tgtcctgccg 1620 ctccgcggta agctctccttttgggaggca ggtactacta aagccgggta cccttataat 1680 tataacacca ctgctagtgaccaactgctc gttgagaatg ccgctgggca tcgggttgct 1740 atttccactt acaccactagcctgggtgct ggtcccgtct ctatttccgc ggttgctgtt 1800 ttagcccccc ctccgcgctagcattgcttg aggataccat ggactaccct gcccgcgccc 1860 atactttcga tgacttctgcccggagtgcc gcccccttgg cctccagggc tgtgcttttc 1920 agtctactgt cgctgagcttcagcgcctta agatgaaggt gggtaaaact cgggagttat 1980 agtttattt 1989 7 169PRT hepatitis E virus 7 Met Val Ile Gln Asp Tyr Asp Asn Gln His Glu GlnAsp Arg Pro Thr 1 5 10 15 Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser ValLeu Arg Ala Asn Asp 20 25 30 Val Leu Trp Leu Ser Leu Thr Ala Ala Glu TyrAsp Gln Ser Thr Tyr 35 40 45 Gly Ser Ser Thr Gly Pro Val Tyr Val Ser AspSer Val Thr Leu Val 50 55 60 Asn Val Ala Thr Gly Ala Gln Ala Val Ala ArgSer Leu Asp Trp Thr 65 70 75 80 Lys Val Thr Leu Asp Gly Arg Pro Leu SerThr Ile Gln Gln Tyr Ser 85 90 95 Lys Thr Phe Phe Val Leu Pro Leu Arg GlyLys Leu Ser Phe Trp Glu 100 105 110 Ala Gly Thr Thr Lys Ala Gly Tyr ProTyr Asn Tyr Asn Thr Thr Ala 115 120 125 Ser Asp Gln Leu Leu Val Glu AsnAla Ala Gly His Arg Val Ala Ile 130 135 140 Ser Thr Tyr Thr Thr Ser LeuGly Ala Gly Pro Val Ser Ile Ser Ala 145 150 155 160 Val Ala Val Leu AlaPro Pro Pro Arg 165 8 159 PRT hepatitis E virus 8 Met Gln Asp Arg ProThr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser 1 5 10 15 Val Leu Arg AlaAsn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu 20 25 30 Tyr Asp Gln SerThr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser 35 40 45 Asp Ser Val ThrLeu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala 50 55 60 Arg Ser Leu AspTrp Thr Lys Val Thr Leu Asp Gly Arg Pro Leu Ser 65 70 75 80 Thr Ile GlnGln Tyr Ser Lys Thr Phe Phe Val Leu Pro Leu Arg Gly 85 90 95 Lys Leu SerPhe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr 100 105 110 Asn TyrAsn Thr Thr Ala Ser Asp Gln Leu Leu Val Glu Asn Ala Ala 115 120 125 GlyHis Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly 130 135 140Pro Val Ser Ile Ser Ala Val Ala Val Leu Ala Pro Pro Pro Arg 145 150 1559 149 PRT hepatitis E virus 9 Met Ser Arg Pro Phe Ser Val Leu Arg AlaAsn Asp Val Leu Trp Leu 1 5 10 15 Ser Leu Thr Ala Ala Glu Tyr Asp GlnSer Thr Tyr Gly Ser Ser Thr 20 25 30 Gly Pro Val Tyr Val Ser Asp Ser ValThr Leu Val Asn Val Ala Thr 35 40 45 Gly Ala Gln Ala Val Ala Arg Ser LeuAsp Trp Thr Lys Val Thr Leu 50 55 60 Asp Gly Arg Pro Leu Ser Thr Ile GlnGln Tyr Ser Lys Thr Phe Phe 65 70 75 80 Val Leu Pro Leu Arg Gly Lys LeuSer Phe Trp Glu Ala Gly Thr Thr 85 90 95 Lys Ala Gly Tyr Pro Tyr Asn TyrAsn Thr Thr Ala Ser Asp Gln Leu 100 105 110 Leu Val Glu Asn Ala Ala GlyHis Arg Val Ala Ile Ser Thr Tyr Thr 115 120 125 Thr Ser Leu Gly Ala GlyPro Val Ser Ile Ser Ala Val Ala Val Leu 130 135 140 Ala Pro Pro Pro Arg145 10 139 PRT hepatitis E virus 10 Met Asp Val Leu Trp Leu Ser Leu ThrAla Ala Glu Tyr Asp Gln Ser 1 5 10 15 Thr Tyr Gly Ser Ser Thr Gly ProVal Tyr Val Ser Asp Ser Val Thr 20 25 30 Leu Val Asn Val Ala Thr Gly AlaGln Ala Val Ala Arg Ser Leu Asp 35 40 45 Trp Thr Lys Val Thr Leu Asp GlyArg Pro Leu Ser Thr Ile Gln Gln 50 55 60 Tyr Ser Lys Thr Phe Phe Val LeuPro Leu Arg Gly Lys Leu Ser Phe 65 70 75 80 Trp Glu Ala Gly Thr Thr LysAla Gly Tyr Pro Tyr Asn Tyr Asn Thr 85 90 95 Thr Ala Ser Asp Gln Leu LeuVal Glu Asn Ala Ala Gly His Arg Val 100 105 110 Ala Ile Ser Thr Tyr ThrThr Ser Leu Gly Ala Gly Pro Val Ser Ile 115 120 125 Ser Ala Val Ala ValLeu Ala Pro Pro Pro Arg 130 135 11 123 PRT hepatitis E virus 11 Met AsnAsn Met Ser Phe Ala Ala Pro Met Gly Ser Arg Pro Cys Ala 1 5 10 15 LeuGly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro 20 25 30 ArgHis Arg Pro Val Ser Arg Leu Ala Ala Val Val Gly Gly Ala Ala 35 40 45 AlaVal Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro 50 55 60 SerGln Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser 65 70 75 80Pro Leu Arg Pro Gly Leu Asp Leu Val Phe Ala Asn Pro Pro Asp His 85 90 95Ser Ala Pro Leu Gly Val Thr Arg Pro Ser Ala Pro Pro Leu Pro His 100 105110 Val Val Asp Leu Pro Gln Leu Gly Pro Arg Arg 115 120 12 232 PRThepatitis E virus 12 Met Ser Lys Ala Phe Ser Asn Cys Tyr Pro Tyr Asp ValPro Asp Tyr 1 5 10 15 Ala Ser Leu Gly Gly Ser Gln Leu Phe Tyr Ser ArgPro Val Val Ser 20 25 30 Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr SerVal Glu Asn Ala 35 40 45 Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp IleAsp Leu Gly Glu 50 55 60 Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln HisGlu Gln Asp Arg 65 70 75 80 Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro PheSer Val Leu Arg Ala 85 90 95 Asn Asp Val Leu Trp Leu Ser Leu Thr Ala AlaGlu Tyr Asp Gln Ser 100 105 110 Thr Tyr Gly Ser Ser Thr Gly Pro Val TyrVal Ser Asp Ser Val Thr 115 120 125 Leu Val Asn Val Ala Thr Gly Ala GlnAla Val Ala Arg Ser Leu Asp 130 135 140 Trp Thr Lys Val Thr Leu Asp GlyArg Pro Leu Ser Thr Ile Gln Gln 145 150 155 160 Tyr Ser Lys Thr Phe PheVal Leu Pro Leu Arg Gly Lys Leu Ser Phe 165 170 175 Trp Glu Ala Gly ThrThr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr 180 185 190 Thr Ala Ser AspGln Leu Leu Val Glu Asn Ala Ala Gly His Arg Val 195 200 205 Ala Ile SerThr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val Ser Ile 210 215 220 Ser AlaVal Ala Val Leu Ala Pro 225 230

What is claimed is
 1. A polypeptide comprising the amino acid sequenceas set forth in SEQ ID No. 1 of hepatitis E virus ORF 2 or its fragment,which is in the form of n-polymeric polypeptide, wherein n is an integerfrom 1-180, said polypeptide comprising the amino acid as set forth inSEQ ID No. 1 of hepatitis E virus ORE 2 or its fragment is selected fromthe group consisting of: 1) A polypeptide having the amino terminusstarts from between amino acid residues 113 and 469, and the carboxylterminus ends from between amino acid residues 596 and 660; 2) Apolypeptide having the amino terminus starts from between amino acidresidues 370 and 469, and the carboxyl terminus ends from between aminoacid residues 601 and 628; 3) A polypeptide having the amino terminusstarts from between amino acid residues 390 and 459, and the carboxylterminus ends from between amino acid residues 601 and 610; 4) Apolypeptide having the amino acid sequence of amino acid residues 414 to660 from SEQ ID NO: 1, i.e., polypeptide 247; 5) A polypeptide havingthe amino acid sequence of amino acid residues 429 to 660 from EQ ID NO:1, i.e., polypeptide 232, 6) A potypeptide having the amino acidsequence of amino acid residues 439 to 660 from SEQ ID NO: 1, i.e.,polypeptide 222; 7) A polypeptide having the amino acid sequence ofamino acid residues 459 to 660 from SEQ ID NO: 1, i.e., polypeptide 201;8) A polypeptide having the amino acid sequence of amino acid residues394 to 628 from SEQ ID NO: 1, i.e., polypeptide 235N; 9) A polypeptidehaving the amino acid sequence of amino acid residues 394 to G18 fromSEQ ID NO: 1, i.e., polypeptide 225N; 10) A polypeptide having the aminoacid sequence of amino acid residues 394 to 602 from SEQ ID NO: 1, i.e.,polypeptide 209N; 11) A polypeptide having the amino acid sequence ofamino acid residues 394 to 601 from SEQ ID NO: 1, i.e., polypeptide208N; 12) A polypeptide having the amino acid sequence of amino acidresidues 394 to 606 from SEQ ID NO: 1, i.e., polypeptide NE21; 13) Apolypeptide having the amino acid sequence of amino acid residues 390 to603 from SEQ ID NO: 1, i.e., polypeptide 217D; 14) A polypeptide havingthe amino acid sequence of amino acid residues 374 to 618 from SEQ IDNO:I, i.e., polypeptide 205; 15) A polypeptide having the amino acidsequence of amino acid residues 414 to 602 from SEQ ID NO: 1, i.e.,polypeptide 189; 16) A polypeptide having the amino acid sequence ofamino acid residues 414 to 601 from SEQ ID NO: 1, i.e., polypeptide 188;17) A polypeptide having the amino acid sequence of amino acid residues459 to 628 from SEQ ID NO: 1; and 18) A polypeptide having the aminoacid sequence of amino acid residues X to 603 from SEQ ID NO: 1 with Metadded at N-terminus and a modified C-terminus, wherein said modifiedC-terminus refers to add, in the direction from 5′- 3′, amino acidsequence-Pro-Pro-Arg at amino acid residue 603, Pro, on its 3′ end;including: a) when X is amino acid residue 394, said polypeptide is asset forth in SEQ ID NO: 2, i.e., NE2; b) when X is amino acid residue414, said polypeptide is as set forth in SEQ ID NO: 3, i.e., 193C; c)when X is amino acid residue 429, said polypeptide is as set forth inSEQ ID NO: 4, i.e., 178C; d) when X is amino acid residue 439, saidpolypeptide is as set forth in SEQ ID NO: 7, i.e., 168C; e) when X isamino acid residue 449, said polypeptide is as set forth in SEQ ID NO:8, i.e., 158C; f) when X is amino acid residue 459, said polypeptide isas set forth in SEQ ID NO: 9, i.e., 148C; g) when X is amino acidresidue 469, said polypeptide is as set forth in SEQ ID NO: 10, i.e.,138C; 19) A polypeptide having at least 80% homology to any one of thepreceding polypeptides of the above 1)- 18) and having substantiallyidentical biological property, such as antigenicity or immunogenicity.2. A recombinant expression vector comprising the nucleotide sequenceencoding polypeptide according to claim
 1. 3. A host cell transformedwith the recombinant expression vector according to claim 2, which isable to express polypeptide according to claim
 1. 4. A vaccinecomposition for prophylaxis and/or treatment of hepatitis E virusinfection in mammals, which comprises at least one of the polypeptidesaccording to claim 1 or any combination thereof, and optionally,pharmaceutically acceptable vehicles and/or adjuvant.
 5. A chimericprotein comprising a polypeptide according to claim 1 and a conservedfragment of hemagglutin antigen from influenza virus.
 6. A vaccinecomposition for prophylaxis and/or treatment of hepatitis E virusinfection in mammals, which comprises chimeric protein according toclaim 5, and optionally, pharmaceutically acceptable vehicles and/oradjuvant.
 7. A polypeptide according to claim 1 or chimeric proteinaccording to claim 5 for vaccinating mammals to prevent from hepatitis Evirus infection.
 8. Use of vaccine composition according to claim 4 or 6for vaccinating mammals to prevent from hepatitis E virus infection. 9.A method for prophylaxis and/or treatment of hepatitis E virus infectionin mammals, which comprises administrating to the subject with aprophylaxis and/or treatment effective amount of polypeptide (s)according to claim 1 or chimeric protein (s) according to claim
 5. 10. Adiagnostic kit for the diagnosis of hepatitis X virus infection inbiological sample, which comprises a diagnosis effective amount of atleast one of the polypeptides according to claim 1 or any combinationthereof
 11. The diagnostic kit according to claim 10, which furthercomprises the polypeptide containing immunogenic epitope from hepatitisE virus ORF3 or an immunogenic fragment thereof, wherein saidpolypeptide containing immunogenic epitope from hepatitis E virus ORF3or an immunogenic fragment thereof is, optionally, covalently bound tothe selected polypeptide according to claim
 1. 12. The diagnostic kitaccording to claim 10 or 11 for determination of antibody IgG againsthepatitis B virus in the biological sample, which comprises at least oneof the polypeptides according to claim 1, if desired, said polypeptideis pre-coated on the surface of a suitable support; and furthercomprises detectable labeled antibody anti-IgG that is directed againstIgG from biological sample to be detected, and detection agentcorresponding to said detectable label; and if desired, furthercomprises a suitable buffer system.
 13. The diagnostic kit according toclaim 10 or 11 for determination of antibody IgM against hepatitis Evirus in the biological sample, which comprises detectable labeledantibody anti-IgM as capture antibody that is directed against IgM frombiological sample to be detected, if desired, said capture antibody ispre-coated on the surface of a suitable support; and further comprisesat least one of the detectable labeled polypeptides according to claim1, and detection agent corresponding to said detectable label; ifdesired, further comprises a suitable buffer system.
 14. The diagnostickit according to claim 10 or 11 for determination of total antibodiesagainst hepatitis E virus in the biological sample, which comprises atleast one of the polypeptides according to claim 1, if desired, saidpolypeptide is pre-coated on the surface of a suitable support; andfurther comprises at least one of the detectable labeled polypeptidesaccording to claim 1, and detection agent corresponding to saiddetectable label; wherein said polypeptide selected from polypeptidesaccording to claim 1 for pre-coating the surface of a support and thedetectable labeled polypeptide selected from polypeptides according toclaim 1 could be the same polypeptide, or different one.
 15. A methodfor diagnosis hepatitis E virus infection in mammals, comprisingcontacting the diagnostic kit according to claim 10 or 11 with sample ofmammal to be detected under the conditions suitable for the interactionof antigen and antibody.
 16. A method for detecting total antibodiesagainst hepatitis E virus in biological samples, comprising the step of:immobilizing at least one of the polypeptides according to claim 1 onthe surface of a support; contacting it with sample to be detected underthe conditions suitable for the interaction of antigen and antibody;washing with a suitable buffer; and detecting antigen/antibody complexon the surface of a support by using antigen of hepatitis E virus with adetectable label and corresponding detect agent.
 17. A method fordetecting antibody IgG against hepatitis E virus in biological samples,comprising the step of: immobilizing at least one of the polypeptidesaccording to claim 1 on the surface of a support; contacting it withsample to be detected under the conditions suitable for the interactionof antigen and antibody, washing with a suitable buffer; and detectingthe antigen/antibody complex on the surface of a support by usingdetectable labeled antibody anti-IgG against hepatitis E virus andcorresponding detect agent.
 18. A method for detecting antibody IgMagainst hepatitis E virus in biological samples, comprising the step of:immobilizing antibody anti-IgM on the surface of a support; contactingit with sample to be detected under the conditions suitable for theinteraction of antigen and antibody; washing with a suitable buffer; anddetecting the anti-IgM/IgM complex on the surface of a support by usingdetectable labeled at least one of the polypeptides according to claim 1and corresponding detect agent.
 19. A method for detecting antibody IgMagainst hepatitis E virus in biological samples, comprising the step of:immobilizing antibody anti-IgM on the surface of a support; contactingit with sample to be detected under conditions suitable for theinteraction of antigen and antibody; washing with a suitable buffer; andthen contacting with at least one of polypeptides according to claim 1under conditions suitable for the interaction of antigen and antibody;washing with a suitable buffer; and then detecting antigen/antibodycomplex on the surface of a support by using detectable labeled anti-HEVpolyclonal or monoclonal antibody and corresponding detect agent.